U.S. patent number 11,267,871 [Application Number 15/320,501] was granted by the patent office on 2022-03-08 for topical application for an anti-hsv antibody.
This patent grant is currently assigned to Heiselberg ImmunoTherapeutics GmbH. The grantee listed for this patent is Heidelberg ImmunoTherapeutics GmbH. Invention is credited to Michaela Arndt, Dirk Jager, Jurgen Krauss.
United States Patent |
11,267,871 |
Arndt , et al. |
March 8, 2022 |
Topical application for an anti-HSV antibody
Abstract
Described is an anti-HSV antibody or an antigen-binding fragment
thereof for use in treating an acute infection of mucosal or
epidermal tissue in a subject caused by HSV-1 or HSV-2 selected
from the group consisting of Herpes simplex labialis, Herpes
simplex genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum, wherein said
antibody is to be topically administered as well as to a
pharmaceutical composition comprising an effective amount of said
antibody or antigen-binding fragment thereof and at least one
pharmaceutically acceptable excipient.
Inventors: |
Arndt; Michaela (Mannheim,
DE), Krauss; Jurgen (Mannheim, DE), Jager;
Dirk (Heidelberg, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Heidelberg ImmunoTherapeutics GmbH |
Heidelberg |
N/A |
DE |
|
|
Assignee: |
Heiselberg ImmunoTherapeutics
GmbH (Heidelberg, DE)
|
Family
ID: |
1000006157912 |
Appl.
No.: |
15/320,501 |
Filed: |
June 25, 2015 |
PCT
Filed: |
June 25, 2015 |
PCT No.: |
PCT/EP2015/064378 |
371(c)(1),(2),(4) Date: |
December 20, 2016 |
PCT
Pub. No.: |
WO2015/197763 |
PCT
Pub. Date: |
December 30, 2015 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20170152304 A1 |
Jun 1, 2017 |
|
Foreign Application Priority Data
|
|
|
|
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Jun 26, 2014 [EP] |
|
|
14174174 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
45/06 (20130101); C07K 16/087 (20130101); A61K
39/42 (20130101); C07K 2317/565 (20130101); A61K
2039/54 (20130101); C07K 2317/24 (20130101); A61K
2039/505 (20130101); C07K 2317/56 (20130101); C07K
2317/76 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); A61K 39/245 (20060101); A61K
45/06 (20060101); C07K 16/08 (20060101); A61K
39/42 (20060101); A61K 39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
|
2308895 |
|
Apr 2011 |
|
EP |
|
2005023303 |
|
Mar 2005 |
|
WO |
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WO 2005/023303 |
|
Mar 2005 |
|
WO |
|
2010128053 |
|
Nov 2010 |
|
WO |
|
2010129033 |
|
Nov 2010 |
|
WO |
|
2011038933 |
|
Apr 2011 |
|
WO |
|
Other References
Krawczyk et al. Overcoming drug-resistant herpes simplex virus
(HSV) infection by a humanized antibody. PNAS, 110(17): 6760-6765,
Apr. 23, 2013. cited by examiner .
Moser et al. Correlation of Herpes Simplex Virus Antibody Titers
and Specific Lymphocyte Stimulation in Adult Blood Donors. Journal
of Clinical Microbiology, Jan. 1981, 13: 36-41. cited by examiner
.
Cocchi et al. Cell-to-Cell Spread of Wild-Type Herpes Simplex Virus
Type 1, but Not of Syncytial Strains, Is Mediated by the
Immunoglobulin-Like Receptors That Mediate Virion Entry, Nectin1
(PRR1/HveC/HlgR) and Nectin2 (PRR2/HveB). J. Virol. 2000,
74:3909-3917. cited by examiner .
De Logu et al. Characterization of a Type-Common Human Recombinant
Monoclonal Antibody to Herpes Simplex Virus with High Therapeutic
Potential. J. Clin. Microbiol. 1998, 36: 3198-3204. cited by
examiner .
Wald et al. Chapter 36 Persistence in the population: epidemiology,
transmission. In Arvin et al., editors. Human Herpesviruses:
Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge
University Press; 2007. cited by examiner .
Emmert et al. Treatment of Common Cutaneous Herpes Simplex Virus
Infections. Am Fam Physician. Mar. 15, 2000;61 (6):1697-1704. cited
by examiner .
International Preliminary Report on Patentability and Written
Opinion for PCT/EP2015/064378 dated Jan. 5, 2017. cited by
applicant .
Extended European Search Report from corresponding EP 14 17 4174,
dated Jan. 16, 2015. cited by applicant .
Krawczyk et al., "Overcoming Drug-Resistant Herpes Simplex Virus
(HSV) Infection by a Humanized Antibody", Proceedings of the
National Academy of Sciences, vol. 110, No. 17, Apr. 8, 2013,
XP055160621. cited by applicant .
Daumer et al., "Characterisation of the Epitope for a Herpes
Simplex Virus Glycoprotein B-specific Monoclonal Antibody with High
Protective Capacity", Medical Microbiology and Immunology, vol.
200, No. 2, Oct. 8, 2010, pp. 85-97, XP055160623. cited by
applicant .
International Search Report & Written Opinion from
corresponding PCT/EP2015/064378, dated Sep. 17, 2015. cited by
applicant .
European Office Action dated Jan. 2, 2018 and received in
15733409.5. cited by applicant .
Sherwood, et al., "Controlled Release of Antibodies For Long-Term
Topic Passive Immunoprotection Of Female Mice Against Genital
Herpes", Nature Biotechnology, vol. 14, Apr. 14, 1996, pp. 468-471
(XP055436671). cited by applicant .
Office of Action dated Jul. 23, 2018 received in corresponding
Russian Patent Application No. 2017 102 387 and translation. cited
by applicant .
Japanese Office action and translation dated Jan. 24, 2019 and
received for corresponding JP Patent Application No. 2016-575171.
cited by applicant .
Baumgarth et al., "B-1 and B-2 Cell-derived Immunoglobulin M
Antibodies Are Nonredundant Components of the Protective Response
to Influenza Virus Infection", J. Exp. Med., vol. 192, No. 2, pp.
271-280, (2000). cited by applicant .
Briles et al., "Antiphosphocholine Antibodies Found in Normal Mouse
Serum are Protective Against Intravenousinfection With Type 3
Streptococcus pneumoniae", J. Exp. Med., vol. 153, pp. 694-705,
(1981). cited by applicant .
Desphande et al., "Dual Role of B Cells in Mediating Innate and
Acquired Immunity to Herpes Simplex Virus Infections", Cellular
Immunology, vol. 202, pp. 79-87, (2000). cited by applicant .
Gobet et al., "The Role of Antibodies in Natural and Acquired
Resistance of Mice to Vesicular Stomatitis Virus", Expl Cell Biol,
vol. 56, pp. 175-180, (1988). cited by applicant .
Haury et al., "The Repertoire of Serum IgM in Normal Mice is
Largely Independent of External Antigenic Contact", Eur.J.
Immunol., vol. 27, pp. 1557-1563, (1997). cited by applicant .
Ochsenbein et al., "Control of Early Viral and Bacterial
Distribution and Disease by Natural Antibodies", Science , vol.
286, pp. 2156-2159, (1999). cited by applicant.
|
Primary Examiner: Zou; Nianxiang
Attorney, Agent or Firm: Wales; Michele M. InHouse Patent
Counsel, LLC
Claims
The invention claimed is:
1. A method of treating an acute infection of mucosal or epidermal
tissue in a subject caused by HSV-1 or HSV-2, wherein the method
comprises administering to the subject a neutralizing full-length
anti-HSV antibody, wherein the subject is suffering from an acute
infection of one or more of Herpes simplex labialis, Herpes simplex
genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum or Eczema herpeticum, wherein the
antibody comprises complementarity determining regions V.sub.HCDR1
comprising SEQ ID NO: 1, V.sub.HCDR2 comprising SEQ ID NO: 2,
V.sub.HCDR3 comprising SEQ ID NO: 3, V.sub.LCDR1 comprising SEQ ID
NO: 4, V.sub.LCDR2 comprising SEQ ID NO: 5, and V.sub.LCDR3
comprising SEQ ID NO:6, wherein said antibody inhibits cell-to-cell
spread, and wherein said antibody is topically administered to
mucosal or epidermal tissue.
2. The method of claim 1, wherein said anti-HSV antibody is a
monoclonal or a polyclonal antibody.
3. The method of claim 1, wherein said anti-HSV antibody is a
humanized or fully human antibody.
4. The method of claim 1, wherein said anti-HSV antibody recognizes
the glycoprotein B (gB) of the HSV-1 and/or HSV-2.
5. The method of claim 1, wherein the antibody is capable of
inhibiting cell-to-cell spread independent from antibody-dependent
cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC).
6. The method of claim 1, wherein the antibody is capable of
inhibiting the spreading of HSV from an infected cell to an
adjacent second non-infected cell (cell to cell spread).
7. The method of claim 1, wherein the antibody comprises an amino
acid sequence with at least 70% sequence identity to the amino acid
residues shown in positions 1 to 30, 38 to 51, 68 to 99, and 112 to
122 of SEQ ID NO: 7 and in positions 1 to 23, 41 to 55, 63 to 94,
and 104 to 114 of SEQ ID NO: 8.
8. The method of claim 1, wherein said antibody comprises the VH of
SEQ ID NO:9 and the VL of SEQ ID NO:10.
9. The method of claim 1, wherein said antibody recognizes the same
epitope as mAb 2c, wherein said epitope is located at the amino
acids 172-195 and 295-313 of glycoprotein B of HSV-1 and HSV-2.
10. The method of claim 1, wherein said antibody is the mAb 2c
antibody.
11. The method of claim 1, wherein said antibody is to be topically
applied to infected mucosal or epidermal tissue of the lips,
genitals, nose, ears, eyes, fingers, toes and/or skin areas
throughout the body, preferably on the head, the jaw area, neck,
chest, face, stomach and/or legs.
12. The method of claim 1, wherein said antibody is to be topically
applied to areas surrounding the infected mucosal or epidermal
tissue.
13. The method of claim 1, wherein said antibody is to be
administered in combination with a virostatic agent.
14. The method of claim 13, wherein said virostatic agent is
selected from the group consisting of the drug classes of
nucleoside analogues, pyrophosphate analogues, nucleotide
analogues, an amantadin derivative, and helicase-primase
inhibitors.
15. The method of claim 14, wherein said nucleoside analogue is
selected from the group consisting of Acyclovir, Penciclovir,
Valacyclovir and Famaciclovir; wherein said pyrophosphate analogue
is Foscarnet; wherein said nucleotide analogue is Cidofovir;
wherein said amantadin derivative is Tromantandin; and wherein said
helicase-primase inhibitor is Pritelevir.
16. The method of claim 1, wherein the antibody is part of a
pharmaceutical composition comprising at least one pharmaceutically
acceptable excipient.
17. The method of claim 1, wherein said anti-HSV antibody comprises
SEQ ID NO:9.
18. The method of claim 1, wherein said anti-HSV antibody comprises
SEQ ID NO:10.
19. The method of claim 1, wherein symptoms of said acute infection
include itching of the lips, burning or tingling near the lips or
mouth area, or blisters.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a .sctn. 371 National Stage Application of
PCT/EP2015/064378, filed 25 Jun. 2015, which claims priority to EP
14174174.4, filed 26 Jun. 2014. These documents (PCT/EP2015/064378
and EP 14174174.4) are hereby incorporated by reference in their
entirety.
The present invention relates to an anti-HSV antibody or an
antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically administered
as well as to a pharmaceutical composition comprising an effective
amount of said antibody or antigen-binding fragment thereof and at
least one pharmaceutically acceptable excipient.
Herpes simplex virus (HSV) refers to two closely related members of
the herpesviridae family, Herpes simplex virus type 1 (HSV-1) and
Herpes simplex virus type 2 (HSV-2). HSV-1 and HSV-2 are among the
most common viral infections in the world. HSV-1 infections are
often acquired in early childhood as subclinical infections while a
subset present with severe disease. HSV-2 is usually acquired
through sexual activity and mainly causes lesions in the genital
area. Infection with the herpes virus is categorized into one of
several distinct disorders based on the site of infection. Oral
herpes (Herpes simplex labialis), the visible symptoms of which are
colloquially called cold sores or fever blisters, is an infection
of the face or mouth. Oral herpes is the most common form of
infection. Genital herpes (Herpes simplex genitalis) is the second
most common form of herpes. Other disorders such as herpetic
whitlow, herpes gladiatorum, ocular herpes (Herpes simplex conreae
or Herpes simplex Keratitis), cerebral herpes infection
encephalitis, Mollaret's meningitis, neonatal herpes, and possibly
Bell's palsy are all caused by herpes simplex viruses.
After primary infection HSV spreads from infected epithelial cells
to axons of sensory neurons innervating the site of the primary
infection followed by retrograde transport to the respective dorsal
root ganglia, where HSV establishes a latent reservoir for life.
HSV infection of neurons exists as a reversible state and episodes
of viral reactivation (outbreaks) may occur from time to time.
Reactivation of the virus can be triggered by a wide range of
stress stimuli (e.g. immunodeficiency, trauma, fever, menstruation,
UV light and sexual intercourse) that act on the neuron, or at a
peripheral site innervated by the infected ganglion, or
systemically. Intermittent HSV reactivations result in the
production of infectious HSV from latently infected neurons. Once
reactivated the virus is transported by the neuron back to the
nerve terminals in the epithelium.
The pathology of HSV infections is mainly caused by a direct
cytopathic effect of the virus, resulting in cellular lysis and
focal necrosis of the infected area. Herpes simplex is most easily
transmitted by direct contact with a lesion or the body fluid of an
infected individual. Oral herpes is easily diagnosed if the patient
presents with visible sores or ulcers. Transmission may also occur
through skin-to-skin contact during periods of asymptomatic
shedding. Although most individuals infected with genital herpes
are asymptomatic, severe clinical manifestations, especially in
populations with underlying immune compromising conditions, can
occur. HSV-2 increases the risk of HIV acquisition by two to
three-fold as well as HIV transmission in dually infected
individuals. In addition, genital herpes can be perinatally
transmitted and cause life-threatening neonatal HSV infection.
Barrier protection methods are the most reliable method of
preventing transmission of herpes, but they merely reduce rather
than eliminate risk.
A cure for herpes has not yet been developed. Once infected, the
virus remains in the body for life. Recurrent infections
(outbreaks) may occur from time to time. However, after several
years, outbreaks become less severe and more sporadic, and some
people will become perpetually asymptomatic and will no longer
experience outbreaks, though they may still be contagious to
others. Treatments with antivirals can reduce viral shedding and
alleviate the severity of symptomatic episodes.
Herpes simplex labialis (also called cold sores, herpes simplex
labialis, recurrent herpes labialis, or orolabial herpes) is a type
of herpes simplex occurring on the lip, i.e., an infection caused
by herpes simplex virus (HSV). An outbreak typically causes small
blisters or sores on or around the mouth commonly known as cold
sores or fever blisters. The sores typically heal within 2 to 3
weeks, but the herpes virus remains dormant in the facial nerves,
following orofacial infection, periodically reactivating (in
symptomatic people) to create sores in the same area of the mouth
or face at the site of the original infection. Cold sore has a rate
of frequency that varies from rare episodes to 12 or more
recurrences per year. People with the condition typically
experience one to three attacks annually. The frequency and
severity of outbreaks generally decreases over time.
Herpes simplex genitalis (or genital herpes) is a genital infection
caused by the herpes simplex virus. A 1998 study indicated it was
the most common sexually transmitted infection by the number of
cases. Most individuals carrying herpes are unaware they have been
infected and many will never suffer an outbreak, which involves
blisters similar to cold sores. While there is no cure for herpes,
over time symptoms are increasingly mild and outbreaks are
decreasingly frequent. As mentioned, HSV has been classified into
two distinct categories, HSV-1 and HSV-2. Although genital herpes
was previously caused primarily by HSV-2, genital HSV-1 infections
are increasing and now cause up to 80% of infections. When
symptomatic, the typical manifestation of a primary HSV-1 or HSV-2
genital infection is clusters of genital sores consisting of
inflamed papules and vesicles on the outer surface of the genitals,
resembling cold sores. These usually appear 4-7 days after sexual
exposure to HSV for the first time. Genital HSV-1 infection recurs
at rate of about one sixth of that of genital HSV-2.
Herpetic simplex keratitis is an inflammation of the eye
predominantly caused by recurrent HSV infection of the cornea.
Ocular infection with HSV can cause eye disease of different
severity, ranging from conjunctivitis and dendritic keratitis to
stromal edema and necrotizing stromal keratitis. HSV-1 causes more
than 90% of ocular HSV infections and is the leading cause of
viral-induced blindness in developed countries.
Moreover, there are other, rather rare HSV infections of mucosal or
epidermal tissue which will be briefly addressed in the
following.
Chronic or disseminated cutaneous herpes simplex infections are
known which are not restricted to labial or genital tract. Mostly,
immunodeficient patients are affected with this disease like, e.g.,
patients with Hypogammaglobulinema or patients with cutaneous
T-cell lymphomas. Chronic cutaneous herpes simplex is a distinctive
clinical presentation of the herpes simplex virus (HSV) in a
compromised host. This infection can be defined as chronically
active destructive skin lesions that potentially may progress into
the disseminated (systemic) form. While most HSV infections display
episodes that show healing in one or two weeks, the lesions of
chronic cutaneous herpes simplex have an indolent course that may
last for several months. Chronic cutaneous herpes simplex, which is
common in immunosuppressed patients, is characterized by atypical,
chronic, and persistent lesions, which complicate and delay the
diagnosis. This may lead to death caused by associated
complications. It is of vital importance that when evaluating
chronic ulcers of long duration, especially in children, the
possibility of a chronic herpes simplex virus infection be
considered. Herpes gladiatorum refers to a herpes skin infection
that occurs in adolescence among wrestlers but it is also common in
other contact sports. It usually occurs on the head, most commonly
the jaw area, the neck, chest, face, stomach, and legs. Eczema
herpeticum, also known as a form of Kaposi varicelliform eruption
caused by viral infection, usually with the herpes simplex virus
(HSV), is an extensive cutaneous vesicular eruption that arises
from pre-existing skin disease, usually atopic dermatitis (AD).
Children with AD have a higher risk of developing eczema
herpeticum, in which HSV type 1 (HSV-1) is the most common
pathogen. Eczema herpeticum can be severe, progressing to
disseminated infection and death if untreated.
Diseases caused by HSV, in particular Herpes simplex labialis and
Herpes simplex genitalis represent the most common infectious
diseases of the skin.
At present, it is standard to use virustatic agents in antiviral
HSV therapy. The most common virustatic agents (e.g., aciclovir,
penciclovir, foscarnet, idoxuridin) are nucleoside or pyrophosphate
analogues whose common active principle is based on the inhibition
of DNA synthesis in virus-infected cells. In other words, these
virustatic agents are only effective in infected cells while the
virus is actively replicating. In a double blinded
placebo-controlled study with 1385 patients suffering from acute
Herpes simplex labialis infection, it has been demonstrated that
Aciclovir (in the form of Zovirax Creme) is capable of reducing the
infection by 0.5 days (i.e., from 5 days to 4.5 days) upon 5.times.
daily administration for 4 days compared to placebo-treated
patients. Moreover, such a treatment suffers from the disadvantage
that the development of lesions which are typical for Herpes cannot
be prevented.
Recently, a murine and a correspondingly humanized antibody has
been described which specifically recognizes the glycoprotein B
(gB) of HSV type 1 (HSV-1) and HSV-2. HSV-gB is an integral part of
the multicomponent fusion system required for virus entry and
cell-cell fusion. This antibody, the monoclonal antibody MAb 2c,
has been demonstrated to neutralize the virus by abrogating viral
cell-to-cell spread, a key mechanism by which HSV-1/2 escapes
humoral immune surveillance independent from antibody-dependent
cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC); Eis-Hubinger et al., Intervirology 32:351-360
(1991); Eis-Hubinger et al., Journal of General Virology 74:379-385
(1993); WO2011/038933 A2; Krawczyk A, et al., Journal of virology
(2011); 85(4):1793-1803; Krawczyk A, et al., Proc Natl Acad Sci USA
(2013); 110(17):6760-6765.
However, these antibodies have only been administered systemically
(i.e., by, e.g., intravenous, intramuscular or subcutaneous
administration) in line with the rationale excluding oral
administration of antibodies due to their size, hydrophilic nature
and degradation in the stomach. Thus, the prior art antibodies have
not been administered locally on the surface of the skin but only
systematically.
WO 2005/023303 discloses a method for the treatment of HSV by an
intravenous administration of IgA fractions of human serum or IgG
fractions of human plasma while a topical administration as well as
the diseases herpes labialis and herpes genitalis are
mentioned.
Although topical administration of antibodies has previously been
described, such an application has only been suggested for
prophylactic use in the prevention of sexually transmitted HSV-2
diseases. Sherwood et al., Nat. Biotechnol. 14(4):468-471 (1996)
describe the prophylactic topical passive immunoprotection of
female mice against genital herpes in a mouse model of
vaginally-transmitted HSV-2 infection by a monoclonal antibody to
HSV-2. Similarly, Zeitlin et al., Virology 225(1):213-215 (1996),
Zeitlin et al., Contraception 56(5):329-335 (1997), Zeitlin et al.,
J. Reprod. Immunol. 40(1):93-101 (1998) and Zeitlin et al., Nat.
Biotechnol. 16(13):1361-1364 (1998) describe the prophylactic
topical administration of anti-HSV-2 antibodies in the prevention
of sexually transmission of HSV-2.
Moreover, the TNF-alpha antibody infliximab has previously been
described to improve the healing of chronic wounds upon topical
application (Streit et al., International Wound Journal
3(3):171-179 (2006)) while the topical application of polyclonal
and monoclonal antibodies against Pseudomonas aeruginosa has
previously been described (U.S. Pat. No. 4,994,269). Furthermore,
Clement et al., ARVO, Abstract/Poster 6155/D1015 describes the
topical administration of an antibody targeting phosphatidylserine
(PS) in a rabbit model of acute HSV-1 Keratitis while Yu et al.,
Eye Science 12(3):145-150 (1996) describe the topical use of
anti-HSV monoclonal glycoprotein antibodies in acute herpetic
Keratitis of rabbits infected by HSV-1. WO 2010/128053 describes
the use of an antibody fragment binding to the viral surface
antigen glycoprotein D neutralizing HSV-1 and HSV-2 for ocular
topical administration for treating ocular diseases like ocular
keratitis.
Thus, there is a need to provide improved means and methods for the
treatment of acute Herpes simplex infections which facilitates
administration regimens known in the art and prevents local
spreading of the infection.
The present invention provides an anti-HSV antibody or an
antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically administered
as well as to a pharmaceutical composition comprising an effective
amount of said antibody or antigen-binding fragment thereof and at
least one pharmaceutically acceptable excipient.
Surprisingly, the present invention demonstrates that the topical
administration of a humanized anti-HSV antibody in an acute
infection of the tissue of the lips upon HSV-infection eliminates
the infection within 24 hours while the local spreading of the
Herpes infection via cell-to-cell spread is prevented, thereby
avoiding the generation of lesions. In contrast to the
above-described virustatic agents used in the treatment of viral
infections like Herpes simplex labialis the anti-HSV antibody of
the present invention is capable of rapidly neutralizing the virus
by a mechanism which is independent of viral replication.
Beneficially, the antibody of the invention is demonstrated to
suppress the lytic route of the virus, thereby preventing skin
lesions.
In view of the prior art, the technical problem underlying the
present invention is the provision of improved means and methods
for the treatment of acute Herpes simplex infections which
facilitates administration regimens known in the art and prevents
local spreading of the infection.
The technical problem is solved by provision of the embodiments
characterized in the claims.
The present invention relates to an anti-HSV antibody or an
antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically
administered.
As mentioned above, it has surprisingly been demonstrated in the
appended examples that the topical administration of a humanized
anti-HSV antibody in an acute infection of the tissue of the lips
upon HSV-infection rapidly eliminates the infection within 24 hours
while the local spreading of the Herpes infection via cell-to-cell
spread is prevented, thereby avoiding the generation of
lesions.
This finding is in particular surprising and unexpected in light of
the prior art discussed above relating to the systemic
administration as described in Eis-Hubinger et al., Intervirology
32:351-360 (1991); Eis-Hubinger et al., Journal of General Virology
74:379-385 (1993); WO2011/038933 A2; Krawczyk A, et al., Journal of
virology (2011); 85(4):1793-1803; Krawczyk A, et al., Proc Natl
Acad Sci USA (2013); 110(17):6760-6765; the prophylactic treatment
as described in Sherwood et al., Nat. Biotechnol. 14(4):468-471
(1996); Zeitlin et al., Virology 225(1):213-215 (1996); Zeitlin et
al., Contraception 56(5):329-335 (1997); Zeitlin et al., J. Reprod.
Immunol. 40(1):93-101 (1998) and Zeitlin et al., Nat. Biotechnol.
16(13):1361-1364 (1998); and the topical treatment with an anti-HSV
glycoprotein antibody in acute Herpes simplex keratitis infection
as described in Yu et al., Eye Science 12(3):145-150 (1996) (and
Clement et al., ARVO, Abstract/Poster 6155/D1015; as well as WO
2010/128053) for the following reasons.
In contrast to the prior art, it has been demonstrated that the
topical administration of a humanized anti-HSV antibody in an acute
infection of the tissue of the lips upon HSV-infection rapidly
eliminates the infection within 24 hours while the local spreading
of the Herpes infection via cell-to-cell spread is prevented,
thereby avoiding the generation of lesions.
The surprising nature of this finding, i.e., that a humanized
anti-HSV antibody or fragment thereof can be used in the topical
therapy of recurrent HSV infections of epithelia of mucosa or skin
is, in particular surprising and unexpected taking into account
basic knowledge about the epidermis structure.
The layers of human skin epithelium and mucous membrane epithelia
physically separate the organism from its environment and serve as
its first line of structural and functional defense against
dehydration, chemical substances, physical insults and
micro-organisms. Highly polarized epithelial cells form the apical
layers of the epidermis and less differentiated cells the basal
region, where the epidermal progenitor cells reside. Occluding
junctions, so called tight junctions (TJ), located at the lateral
plasma membranes of the most superficial living layer, the stratum
granulosum (Brandner, et al., 2002 Eur J Cell Biol 81, 253-263;
Furuse et al., 2002, J Cell Biol 156, 1099-1111) secure the
epidermal barrier function between the apical layer (stratum
corneum) and the basolateral layers (stratum spinosum, stratum
basale & lamina basale).
The principal site of HSV replication and progeny virus production
in the skin are the less differentiated, proliferating
keratinocytes of the basal region (stratum basale) (Mingo et al.,
2012, J Virol 86, 7084-7097; Schelhaas et al., 2003, J Gen Virol
84, 2473-2484).
Pathogenesis of primary infection requires that HSV accesses
permissive nucleated cells in the mid- to basal epidermis via
microscopic breaches in the epidermis that occur for instance with
coitus.
Reactivation of the HSV genome from latency within ganglia leads to
transport of newly formed virions traffic down axon microtubules
for release at synaptic terminals at the dermal-epidermal junction
or within the mid-layer of the epidermis (Diefenbach et al., 2008,
Rev Med Virol 18, 35-51). HSV needs to cross the axonal-epithelial
gap for subsequent replication in the basal region of the
epidermis.
Humoral immunity plays an important role in controlling HSV
infection. Circulating serum antibodies, which can bind viral
envelope glycoproteins necessary for viral entry, develop during
infection (Cohen et al., 1984, Journal of virology 49, 102-108). It
has been shown that the presence of maternal serum antibodies
specific to HSV reduces neonatal transmission of HSV-2 (Brown et
al., 1991, N Engl J Med 324, 1247-1252). Neutralizing serum
antibodies are capable of binding virus in the gap between neuron
endings and epithelial cells and limit bidirectional viral transfer
between these tissues (Mikloska et al., 1999, Journal of virology
73, 5934-5944). Evidently, serum antibodies or systemically applied
antibodies limit the extent of HSV infection.
Tight junctions (TJ), however, which are restricted to the stratum
granulosum, form a barrier for larger molecules (Helfrich et al.,
2007, J Invest Dermatol 127, 782-791; Mertens et al., 2005, J Cell
Biol 170, 1029-1037; Yuki et al., 2007, Exp Dermatol 16, 324-330).
Therefore, it was utmost surprising that topical application of a
large molecule like an antibody to the outer skin is able to
eradicate a recurrent HSV infection effectively and prevents
formation of lesions. Thus, for this reason, it was surprising
vis-a-vis a systemic administration, that a local, topical
administration rapidly eliminates the infection as exemplified in
the examples.
Moreover, although the prior art describes the protection of
sexually transmitted primary HSV-2 infection by a topical
prophylactic application of anti-HSV antibodies the treatment of an
acute infection is surprising because in the experimental setting
of the prior art discussed above, anti-HSV antibodies were
topically applied to the vagina before delivering the virus
inoculum. The viral load is getting neutralized like in a
two-dimensional in vitro neutralization assay, where neutralizing
antibodies prevent attachment of free virus particles to target
cells and virus replication actually does not take place. This is
in stark contrast to the treatment of an acute infection which has
surprisingly been shown to rapidly eliminate the infection by the
topical administration of an antibody of the present invention as
exemplified in the examples. Moreover, although the prior art
describes the topical treatment with an anti-HSV glycoprotein
antibody in acute Herpes simplex keratitis, an anti-HSV antibody or
an antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically administered
is surprising for the following reasons. The principal site of HSV
replication and progeny virus production in the skin are the less
differentiated, proliferating keratinocytes of the basal region
(Mingo et al., 2012, J Virol 86, 7084-7097; Schelhaas et al., 2003,
J Gen Virol 84, 2473-2484). It has been shown that tight junctions
(TJ), which are restricted to the stratum granulosum, form a
barrier for larger molecules (Helfrich et al., 2007, J Invest
Dermatol 127, 782-791; Mertens et al., 2005, J Cell Biol 170,
1029-1037; Yuki et al., 2007, Exp Dermatol 16, 324-330). Therefore,
it was utmost surprising that topical application of a large
molecule like an antibody to the outer skin is able to eradicate a
recurrent HSV infection effectively and prevents formation of
lesions.
In contrast to skin epithelium and mucous membrane epithelia the
cornea of the eye is a non-keratinized stratified squamous
epithelium, which is exceedingly thin and consists of fast-growing
and easily regenerated cells. All layers of the eye epithelium are
constantly undergoing mitosis. The corneal epithelium provides a
smooth surface that absorbs oxygen and cell nutrients from tears,
then distributes these nutrients to the rest of the cornea. Another
major difference to skin epithelium and mucous membrane epithelia
is some degree of leakiness of the corneal endothelium, which is
essential for nutrient diffusion. Ulturastructure studies of the
corneal endothelium confirmed that gaps in specific tight junctions
proteins exist and that the tight junctions of the cornea are
"leaky" junctions (Barry et al., 1995, Invest Ophthalmol Vis Sci
36, 1115-1124; Noske et al., 1994, Ger J Ophthalmol 3, 253-257;
Petroll et al., 1999, Curr Eye Res 18, 10-19). The cornea of the
eye is an immunologically privileged site. The ocular surface is
constantly covered by a tear film, which besides largely consisting
of water although contains a number of proteins that have antiviral
activity such as immunoglobulin A antibodies, lysozyme, complement
and amylase. The presence of anti-HSV antibodies has been
demonstrated in tears (Centifanto et al., 1970, Ann NY Acad Sci
173, 649-656; Fox et al., 1986, The British journal of
ophthalmology 70, 584-588; Shani et al., 1985, Curr Eye Res 4,
103-111). Therefore, topical application of recombinant antibodies
may be beneficial for the treatment of ocular herpes infections.
However, for the above reasons, it was unexpected that topical
application of a large molecule like an antibody to the outer skin
is able to eradicate a recurrent HSV infection effectively and
prevents formation of lesions.
The infections of mucosal or epidermal tissue to be treated with
the anti-HSV antibody or an antigen-binding fragment which are
caused by HSV-1 or HSV-2, i.e., the infections selected from the
group consisting of Herpes simplex labialis, Herpes simplex
genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum are well known
to the person skilled in the art and represent well-defined
diseases. As already mentioned above, Herpes simplex labialis (also
called cold sores, herpes simplex labialis, recurrent herpes
labialis, or orolabial herpes) is a type of herpes simplex
occurring on the lip, i.e. an infection by herpes simplex virus
(HSV). An outbreak typically causes small blisters or sores on or
around the mouth commonly known as cold sores or fever blisters.
The sores typically heal within 2 to 3 weeks, but the herpes virus
remains dormant in the facial nerves, following orofacial
infection, periodically reactivating (in symptomatic people) to
create sores in the same area of the mouth or face at the site of
the original infection. Cold sore has a rate of frequency that
varies from rare episodes to 12 or more recurrences per year.
People with the condition typically experience one to three attacks
annually. The frequency and severity of outbreaks generally
decreases over time.
Herpes simplex genitalis (or genital herpes) is a genital infection
caused by the herpes simplex virus. A 1998 study indicated it was
the most common sexually transmitted infection by the number of
cases. Most individuals carrying herpes are unaware they have been
infected and many will never suffer an outbreak, which involves
blisters similar to cold sores. While there is no cure for herpes,
over time symptoms are increasingly mild and outbreaks are
decreasingly frequent. As mentioned, HSV has been classified into
two distinct categories, HSV-1 and HSV-2. Although genital herpes
was previously caused primarily by HSV-2, genital HSV-1 infections
are increasing and now cause up to 80% of infections. When
symptomatic, the typical manifestation of a primary HSV-1 or HSV-2
genital infection is clusters of genital sores consisting of
inflamed papules and vesicles on the outer surface of the genitals,
resembling cold sores. These usually appear 4-7 days after sexual
exposure to HSV for the first time. Genital HSV-1 infection recurs
at rate of about one sixth of that of genital HSV-2.
Chronic or disseminated cutaneous herpes simplex infections are
known which are not restricted to labial or genital tract. Mostly,
immunodeficient patients are affected with this disease like, e.g.,
patients with Hypogammaglobulinema or patients with cutaneous
T-cell lymphomas. Chronic cutaneous herpes simplex is a distinctive
clinical presentation of the herpes simplex virus (HSV) in a
compromised host. This infection can be defined as chronically
active destructive skin lesions that potentially may progress into
the disseminated (systemic) form. While most HSV infections display
episodes that show healing in one or two weeks, the lesions of
chronic cutaneous herpes simplex have an indolent course that may
last for several months. Chronic cutaneous herpes simplex, which is
common in immunosuppressed patients, is characterized by atypical,
chronic, and persistent lesions, which complicate and delay the
diagnosis. This may lead to death caused by associated
complications. It is of vital importance that when evaluating
chronic ulcers of long duration, especially in children, the
possibility of a chronic herpes simplex virus infection be
considered.
Herpes gladiatorum is a herpes skin infection that occurs in
adolescence among wrestlers but it is also common in other contact
sports. It usually occurs on the head, most commonly the jaw area,
the neck, chest, face, stomach, and legs.
Eczema herpeticum, also known as a form of Kaposi varicelliform
eruption caused by viral infection, usually with the herpes simplex
virus (HSV), is an extensive cutaneous vesicular eruption that
arises from pre-existing skin disease, usually atopic dermatitis
(AD). Children with AD have a higher risk of developing eczema
herpeticum, in which HSV type 1 (HSV-1) is the most common
pathogen. Eczema herpeticum can be severe, progressing to
disseminated infection and death if untreated.
The antibody or fragment thereof as used in the context of the
present invention for use in treating an acute infection of mucosal
or epidermal tissue in a subject caused by HSV-1 or HSV-2 is not
particularly limited as long as it is an "anti-HSV antibody or an
antigen-binding fragment thereof". Thus, the antibody may be any
antibody which specifically binds to or specifically recognizes or
interacts with a HSV, i.e., a domain or an antigen of a HSV.
The term "binding to" as used in the context of the present
invention defines a binding (interaction) of at least two
"antigen-interaction-sites" with each other. The term
"antigen-interaction-site" defines, in accordance with the present
invention, a motif of a polypeptide, i.e., a part of the antibody
or antigen-binding fragment of the present invention, which shows
the capacity of specific interaction with a specific antigen or a
specific group of antigens of the HSV. Said binding/interaction is
also understood to define a "specific recognition". The term
"specifically recognizing" means in accordance with this invention
that the antibody is capable of specifically interacting with
and/or binding to at least two amino acids of each of a HSV as
defined herein. Antibodies can recognize, interact and/or bind to
different epitopes on a HSV. This term relates to the specificity
of the antibody molecule, i.e., to its ability to discriminate
between the specific regions of a HSV.
The term "specific interaction" as used in accordance with the
present invention means that the antibody or antigen-binding
fragment thereof of the invention does not or does not essentially
cross-react with (poly) peptides of similar structures.
Accordingly, the antibody or antigen-binding fragment thereof of
the invention specifically binds to/interacts with structures of a
HSV, preferably HSV-1 or HSV-2. Specific examples of such molecules
against which said first and second, Ig-derived domain is directed
are given herein below.
Cross-reactivity of a panel of antibody or antigen-binding fragment
thereof under investigation may be tested, for example, by
assessing binding of said panel of antibody or antigen-binding
fragment thereof under conventional conditions (see, e.g., Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide
of interest as well as to a number of more or less (structurally
and/or functionally) closely related (poly)peptides. Only those
constructs (i.e. antibodies, antigen-binding fragments thereof and
the like) that bind to the certain structure of the HSV, e.g., a
specific epitope or (poly) peptide/protein of the HSV but do not or
do not essentially bind to any of the other epitope or (poly)
peptides of the same HSV, are considered specific for the epitope
or (poly) peptide/protein of interest and selected for further
studies in accordance with the method provided herein. These
methods may comprise, inter alia, binding studies, blocking and
competition studies with structurally and/or functionally closely
related molecules. These binding studies also comprise FACS
analysis, surface plasmon resonance (SPR, e.g. with BIAcore.RTM.),
analytical ultracentrifugation, isothermal titration calorimetry,
fluorescence anisotropy, fluorescence spectroscopy or by
radiolabeled ligand binding assays.
The term "binding to" does not only relate to a linear epitope but
may also relate to a conformational epitope, a structural epitope
or a discontinuous epitope consisting of two regions of the human
target molecules or parts thereof. In the context of this
invention, a conformational epitope is defined by two or more
discrete amino acid sequences separated in the primary sequence
which comes together on the surface of the molecule when the
polypeptide folds to the native protein (Sela, Science 166 (1969),
1365 and Laver, Cell 61 (1990), 553-536). Moreover, the term
"binding to" is interchangeably used in the context of the present
invention with the terms "interacting with" or "recognizing".
Accordingly, specificity can be determined experimentally by
methods known in the art and methods as described herein. Such
methods comprise, but are not limited to Western Blots, ELISA-,
RIA-, ECL-, IRMA-tests and peptide scans.
The treatment of the present invention relates to the treatment of
acute infections. "Acute" in this respect means that the subject
shows symptoms of the disease. In other words, the subject to be
treated is in actual need of a treatment and the term "acute
treatment" in the context of the present invention relates to the
measures taken to actually treat the disease after the onset or the
breakout of the disease. The term "acute" as referred to in the
context of the present invention is opposed to a prophylactic
treatment or preventive treatment, i.e., measures taken for disease
prevention, e.g., in order to prevent the infection and/or the
onset/outbreak of the disease. More specifically, prophylactic
treatment may be understood in a way that it prevents attachment of
free virus particles (from outside the body) to target cells and in
turn prevents virus replication. In contrast, at an acute infection
(which could be a primary or a recurrent infection) progeny virus
have been raced upon HSV replication. Thus, the "acute treatment"
referred to in the present invention does explicitly not relate to
prophylactic or preventive treatment of an infection caused by
HSV-1 or HSV-2.
Mucosal tissue that may display an acute infection refers to
tissues of the mucous membranes which are linings of mostly
endodermal origin, covered in epithelium, which are involved in
absorption and secretion. They line cavities that are exposed to
the external environment and internal organs. They are at several
places contiguous with skin: e.g., at the nostrils, the lips of the
mouth, the eyelids, the ears, the genital area, and the anus.
Epidermal tissue that may display an acute infection refers to
tissues of the epidermis, i.e., the outermost layers of cells in
the skin, which together with the dermis forms the cutis. The
epidermis is a stratified squamous epithelium composed of
proliferating basal and differentiated suprabasal keratinocytes
which acts as the body's major barrier against an inhospitable
environment, by preventing pathogens from entering, making the skin
a natural barrier to infection. It also regulates the amount of
water released from the body into the atmosphere through
transepidermal water loss.
As mentioned, the anti-HSV antibody or an antigen-binding fragment
thereof for use in treating an acute infection of mucosal or
epidermal tissue in a subject caused by HSV-1 or HSV-2 selected
from the group consisting of Herpes simplex labialis, Herpes
simplex genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum is to be
topically administered.
The term "topical administration" in accordance with the present
invention relates to a medication, application or administration
that is applied to body surfaces such as the skin or mucous
membranes to treat the infection referred to above via a large
range of classes of forms of administration, including but not
limited to creams, foams, gels, lotions and ointments. In a
preferred embodiment, topical administration is understood to be
epicutaneous, meaning that the anti-HSV antibody or an
antigen-binding fragment thereof is applied directly to the skin.
Without being bound by theory and to provide some further
non-limiting examples, topical application may also be
inhalational, such as asthma medications, or applied to the surface
of tissues other than the skin, such as eye drops applied to the
conjunctiva, or ear drops placed in the ear, or medications applied
to the surface of a tooth. As a route of administration, topical
administration is contrasted with enteral (in the digestive tract)
and intravascular/intravenous (injected into the circulatory
system). In its broadest sense, a topical effect may be understood
in a way that it relates to, in the pharmacodynamic sense, a local,
rather than systemic, target for a medication.
In a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according the present
invention is a monoclonal or a polyclonal antibody. In a further
preferred embodiment, the anti-HSV antibody or the antigen-binding
fragment thereof for use according to the present invention is a
humanized or a fully human antibody. In a further preferred
embodiment, the anti-HSV antibody or the antigen-binding fragment
thereof for use according to the present invention is a murine
antibody.
The term "monoclonal antibody" as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Monoclonal antibodies are advantageous in that they
may be synthesized by a hybridoma culture, essentially
uncontaminated by other immunoglobulins. The modified "monoclonal"
indicates the character of the antibody as being amongst a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. As mentioned above, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method described by Kohler, Nature 256 (1975), 495.
The term "polyclonal antibody" as used herein, refers to an
antibody which was produced among or in the presence of one or more
other, non-identical antibodies. In general, polyclonal antibodies
are produced from a B-lymphocyte in the presence of several other
B-lymphocytes which produced non-identical antibodies. Usually,
polyclonal antibodies are obtained directly from an immunized
animal.
The term "fully-human antibody" as used herein refers to an
antibody which comprises human immunoglobulin protein sequences
only. A fully human antibody may contain murine carbohydrate chains
if produced in a mouse, in a mouse cell or in a hybridoma derived
from a mouse cell. Similarly, "mouse antibody" or "murine antibody"
refers to an antibody which comprises mouse/murine immunoglobulin
protein sequences only. Alternatively, a "fully-human antibody" may
contain rat carbohydrate chains if produced in a rat, in a rat
cell, in a hybridoma derived from a rat cell. Similarly, the term
"rat antibody" refers to an antibody that comprises rat
immunoglobulin sequences only. Fully-human antibodies may also be
produced, for example, by phage display which is a widely used
screening technology which enables production and screening of
fully human antibodies. Also phage antibodies can be used in
context of this invention. Phage display methods are described, for
example, in U.S. Pat. Nos. 5,403,484, 5,969,108 and 5,885,793.
Another technology which enables development of fully-human
antibodies involves a modification of mouse hybridoma technology.
Mice are made transgenic to contain the human immunoglobulin locus
in exchange for their own mouse genes (see, for example, U.S. Pat.
No. 5,877,397).
The term "chimeric antibodies", refers to an antibody which
comprises a variable region of the present invention fused or
chimerized with an antibody region (e.g., constant region) from
another, human or non-human species (e.g., mouse, horse, rabbit,
dog, cow, chicken).
The term antibody also relates to recombinant human antibodies,
heterologous antibodies and heterohybrid antibodies. The term
"recombinant human antibody" includes all human sequence antibodies
that are prepared, expressed, created or isolated by recombinant
means, such as antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes; antibodies
expressed using a recombinant expression vector transfected into a
host cell, antibodies isolated from a recombinant, combinatorial
human antibody library, or antibodies prepared, expressed, created
or isolated by any other means that involves splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions (if
present) derived from human germline immunoglobulin sequences. Such
antibodies can, however, be subjected to in vitro mutagenesis (or,
when an animal transgenic for human Ig sequences is used, in vivo
somatic mutagenesis) and thus the amino acid sequences of the VH
and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL
sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
A "heterologous antibody" is defined in relation to the transgenic
non-human organism producing such an antibody. This term refers to
an antibody having an amino acid sequence or an encoding nucleic
acid sequence corresponding to that found in an organism not
consisting of the transgenic non-human animal, and generally from a
species other than that of the transgenic non-human animal.
The term "heterohybrid antibody" refers to an antibody having light
and heavy chains of different organismal origins. For example, an
antibody having a human heavy chain associated with a murine light
chain is a heterohybrid antibody. Examples of heterohybrid
antibodies include chimeric and humanized antibodies.
The term antibody also relates to humanized antibodies. "Humanized"
forms of non-human (e.g. murine or rabbit) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of
antibodies) which contain minimal sequence derived from non-human
immunoglobulin. Often, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibody may comprise
residues, which are found neither in the recipient antibody nor in
the imported CDR or framework sequences. These modifications are
made to further refine and optimize antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody may also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see: JonesNature 321 (1986),
522-525; Reichmann Nature 332 (1998), 323-327 and Presta Curr Op
Struct Biol 2 (1992), 593-596.
A popular method for humanization of antibodies involves CDR
grafting, where a functional antigen-binding site from a non-human
`donor` antibody is grafted onto a human `acceptor` antibody. CDR
grafting methods are known in the art and described, for example,
in U.S. Pat. Nos. 5,225,539, 5,693,761 and 6,407,213. Another
related method is the production of humanized antibodies from
transgenic animals that are genetically engineered to contain one
or more humanized immunoglobulin loci which are capable of
undergoing gene rearrangement and gene conversion (see, for
example, U.S. Pat. No. 7,129,084).
Accordingly, in context of the present invention, the term
"antibody" relates to full immunoglobulin molecules as well as to
parts of such immunoglobulin molecules (i.e., "antigen-binding
fragment thereof"). Furthermore, the term relates, as discussed
above, to modified and/or altered antibody molecules. The term also
relates to recombinantly or synthetically generated/synthesized
antibodies. The term also relates to intact antibodies as well as
to antibody fragments thereof, like, separated light and heavy
chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term antibody also
comprises but is not limited to fully-human antibodies, chimeric
antibodies, humanized antibodies, CDR-grafted antibodies and
antibody constructs, like single chain Fvs (scFv) or
antibody-fusion proteins.
In a preferred embodiment, the anti-HSV antibody for use for use in
treating an acute infection of mucosal or epidermal tissue in a
subject caused by HSV-1 or HSV-2 selected from the group consisting
of Herpes simplex labialis, Herpes simplex genitalis, chronic or
disseminated cutaneous herpes simplex infection, Herpes gladiatorum
and Eczema herpeticum, wherein said antibody is to be topically
administered, is a full-length antibody, i.e., to a full
immunoglobulin molecule which is often also referred to as complete
antibody.
"Single-chain Fv" or "scFv" antibody fragments have, in the context
of the invention, the V.sub.H and V.sub.L domains of an antibody,
wherein these domains are present in a single polypeptide chain.
Generally, the scFv polypeptide further comprises a polypeptide
linker between the V.sub.H and V.sub.L domains which enables the
scFv to form the desired structure for antigen binding. Techniques
described for the production of single chain antibodies are
described, e.g., in Pluckthun in The Pharmacology of Monoclonal
Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994),
269-315.
A "Fab fragment" as used herein is comprised of one light chain and
the C.sub.H1 and variable regions of one heavy chain. The heavy
chain of a Fab molecule cannot form a disulfide bond with another
heavy chain molecule.
An "Fc" region contains two heavy chain fragments comprising the
C.sub.H2 and C.sub.H3 domains of an antibody. The two heavy chain
fragments are held together by two or more disulfide bonds and by
hydrophobic interactions of the C.sub.H3 domains.
A "Fab' fragment" contains one light chain and a portion of one
heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
A "F(ab').sub.2 fragment" contains two light chains and two heavy
chains containing a portion of the constant region between the
C.sub.H1 and C.sub.H2 domains, such that an interchain disulfide
bond is formed between the two heavy chains. A F(ab').sub.2
fragment thus is composed of two Fab' fragments that are held
together by a disulfide bond between the two heavy chains.
The "Fv region" comprises the variable regions from both the heavy
and light chains, but lacks the constant regions.
Antibodies, antibody constructs, antibody fragments, antibody
derivatives (all being Ig-derived) to be employed in accordance
with the invention or their corresponding immunoglobulin chain(s)
can be further modified using conventional techniques known in the
art, for example, by using amino acid deletion(s), insertion(s),
substitution(s), addition(s), and/or recombination(s) and/or any
other modification(s) known in the art either alone or in
combination. Methods for introducing such modifications in the DNA
sequence underlying the amino acid sequence of an immunoglobulin
chain are well known to the person skilled in the art; see, e.g.,
Sambrook (1989), loc. cit. The term "Ig-derived domain"
particularly relates to (poly) peptide constructs comprising at
least one CDR. Fragments or derivatives of the recited Ig-derived
domains define (poly) peptides which are parts of the above
antibody molecules and/or which are modified by
chemical/biochemical or molecular biological methods. Corresponding
methods are known in the art and described inter alia in laboratory
manuals (see Sambrook et al., Molecular Cloning: A Laboratory
Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and
3rd edition (2001); Gerhardt et al., Methods for General and
Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology
Methods Manual: The Comprehensive Sourcebook of Techniques;
Academic Press (1997); Golemis, Protein-Protein Interactions: A
Molecular Cloning Manual Cold Spring Harbor Laboratory Press
(2002)).
The antibody or fragment thereof as used in the context of the
present invention for use in treating an acute infection of mucosal
or epidermal tissue in a subject caused by HSV-1 or HSV-2 selected
from the group consisting of Herpes simplex labialis, Herpes
simplex genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum, wherein said
antibody is to be topically administered, is not particularly
limited as long as it is an "anti-HSV antibody or an
antigen-binding fragment thereof". Thus, the antibody may be any
antibody which specifically binds to or specifically recognizes or
interacts with a HSV, i.e., a domain, an antigen, preferably a
surface-antigen of a HSV. The skilled person is readily in a
position to generate such an antibody directed to a given domain
(i.e., an antigen, preferably a surface-antigen of a HSV) and
determine whether a respective antibody is capable of
detecting/binding to a given domain, an antigen, preferably a
surface-antigen of a HSV, preferably HSV-1 and/or HSV-2 based on
the skilled person's common general knowledge and the methods
described above.
In a preferred embodiment, the antibody of the invention binds
to/recognizes the viral antigen glycoproteins D, B, C, H, L, E or I
(i.e., gD, gB, gC, gH, gL, gE, gl) Glycoproteins D, B, C, H, L, E
and I are surface or envelope proteins of HSV-1 and/or HSV-2. These
proteins may not only be found on the surface or in the envelope
structure of HSV-1 and/or HSV-2, i.e., on the surface of released
infectious particles (i.e., the envelope of free virions) but they
may also be present on the surface of infected cells, i.e., on the
surface of cells. Yet, in a more preferred embodiment, the antibody
of the invention binds to/recognizes the viral surface antigen
glycoprotein D, B, C, H, L, E or I (i.e., gD, gB, gC, gH, gL, gE,
or gl) of the HSV-1 and/or HSV-2 envelope. In a preferred
embodiment, the anti-HSV antibody or the antigen-binding fragment
thereof for use according to the present invention recognizes the
surface glycoprotein B (gB) of the HSV-1 and/or HSV-2 envelope,
preferably an epitope thereof. The glycoprotein B of HSV-1 and/or
HSV-2 is well-characterized and, without being bound to specific
sequences, examples sequences of various HSV-1 and HSV-2 strains,
respectively, are shown in SEQ ID NOs:11 to 16. SEQ ID NO:11 shows
the sequence of the glycoprotein B of HSV-1 strain F, SEQ ID NO:12
shows the sequence of the glycoprotein B of HSV-1 strain KOS, SEQ
ID NO:13 shows the sequence of the glycoprotein B of HSV-1 strain
gC-39-R6, SEQ ID NO:14 shows the sequence of the glycoprotein B of
HSV-2 strain HG52, SEQ ID NO:15 shows the sequence of the
glycoprotein B of HSV-2 strain 333 and SEQ ID NO:16 shows the
sequence of the glycoprotein B of HSV-2 strain MMA. A sequence
alignment of these glycoprotein B amino acid sequences shows that
the overall amino acid homology of gB of HSV-1 and HSV-2 is 85%
while the sequences are least conserved at the N- and C-terminal
regions of the protein.
In a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically administered,
is capable of inhibiting the spreading of HSV from an infected cell
to an adjacent second non-infected cell (cell-to-cell spread).
Cell-to-cell spread is the ability of the herpes virus to spread to
an adjacent second non-infected cell without releasing cell-free
particles. Reducing or eliminating the ability of the herpes virus
to spread to an adjacent cell has the beneficial effect that the
generation of lesions is avoided. In order to examine whether an
antibody is capable of inhibiting the spread of HSV from an
infected cell to an adjacent second non-infected cell (cell-to-cell
spread), methods well-known to the person skilled in the art can be
used. As an example, the following assay can be used: Vero cells
grown to confluency on glass cover slips in 24-well tissue culture
plates are infected for 4 h at 37.degree. C. with a constant virus
amount of 400 TCID.sub.50/well. One median tissue culture infective
dose (1 TCID.sub.50) is the mount of a cytopathogenic agent, such
as a virus, that will produce a cytopathic effect in 50% of the
cell cultures inoculated. The virus inoculum is subsequently
removed, the cells washed twice with PBS and further incubated for
2 days at 37.degree. C. in 1 ml DMEM, 2% FCS, Pen/Strep containing
an excess of either different anti-HSV antibodies or polyclonal
anti-HSV control serum in order to prevent viral spreading via the
supernatant. Viral antigens of HSV-infected cells are detected with
a fluorescence labelled polyclonal goat-anti-HSV-serum (BETHYL
Laboratories, Montgomery, Tex. USA, Catalog No. A190-136F, Lot No.
A190-136F-2). Preferably, an antibody is inhibiting cell-to-cell
spread if less than 20% of the adjacent cells are infected,
preferably wherein less than 15%, less than 10%, less than 5%, more
preferably less than 3% and most preferably less than 1% of the
adjacent cells are infected in the above assay.
Cell-to-cell spread may also be assayed as follows: The presence of
neutralizing antibodies does not necessarily prevent cell-to-cell
spread of herpesviridae. To compare antibodies on disruption of
HSV-1 and HSV-2 cell-to-cell spread this particular dissemination
mode can be mimicked in vitro using standard test methods. E.g.: To
infect individual cells, confluent Vero cell monolayers are
initially incubated with either HSV-1 or HSV-2 at low MOI (e.g. 100
TCID.sub.50), respectively. After 4 h of adsorption at 37.degree.
C., the viral inoculum has to be removed. To promote direct
cell-to-cell transmission from individually infected cells but
prevent viral spread through viral particles across the cell
culture supernatant, Vero cell monolayers are treated with an
excess of neutralizing anti-gB antibodies, controls, or medium
alone. After 48 h virus spread can be detected by immunostaining
with a mouse monoclonal antibody specific for a common epitope on
glycoprotein D of HSV-1 and HSV-2 (e.g. Acris Antibodies, San
Diego, Calif., USA) and fluorescence-conjugated secondary antibody.
Immunofluorescence images can be acquired with a fluorescence
microscope at a 100- or 400-fold magnification.
Moreover, in a preferred embodiment, the anti-HSV antibody of the
present invention is capable of neutralizing HSV. "Neutralizing"
herein means that the antibody opsonizes the virus so that it
cannot infect any further cell. An assay for testing whether an
antibody in a concentration of, e.g., at most 20 nM is capable of
neutralizing a defined amount of HSV of, e.g., 100 TCID.sub.50
Eis-Hubinger et al., Intervirology 32:351-360 (1991); Eis-Hubinger
et al., Journal of General Virology 74:379-385 (1993) and in
Examples 1 and 2 of WO2011/038933 A2. Thus, in a preferred
embodiment, the antibody of the invention in a concentration of at
most 20 nM, preferably of at most 16 nM, more preferably of at most
12 nM, such as of at most 10 nm, e.g., at most 8 nM or at most 6
nM, and most preferably of at most 4 nM is capable of neutralizing
a defined amount of HSV of 100 TCID.sub.50 to more than 80%,
preferably by more than 90%, such as more than 95%, more preferably
96%, e.g., more than 97%, and most preferably more than 98%, e.g.,
more than 99% or even 100%.
Thus, in a preferred embodiment, the present invention also relates
to an anti-HSV antibody or the antigen-binding fragment thereof for
use in treating an acute infection of mucosal or epidermal tissue
in a subject caused by HSV-1 or HSV-2 selected from the group
consisting of Herpes simplex labialis, Herpes simplex genitalis,
chronic or disseminated cutaneous herpes simplex infection, Herpes
gladiatorum and Eczema herpeticum, wherein said antibody is to be
topically administered, wherein the antibody is capable of
inhibiting cell-to-cell spread independent from antibody-dependent
cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC).
As the above-described assays for testing the capability whether an
antibody is capable of inhibiting cell-to-cell spread do not
contain complement and/or cytotoxic effector cells, the same assays
may be used in order to determine whether an antibody is capable of
inhibiting cell-to-cell spread independent from antibody-dependent
cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC).
In a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention comprises the complementarity determining regions
V.sub.HCDR1 comprising SEQ ID NO: 1, V.sub.HCDR2 comprising SEQ ID
NO: 2, V.sub.HCDR3 comprising SEQ ID NO: 3, V.sub.LCDR1 comprising
SEQ ID NO: 4, V.sub.LCDR2 comprising SEQ ID NO: 5, and V.sub.LCDR3
comprising SEQ ID NO:6.
The term "CDR" as employed herein relates to "complementary
determining region", which is well known in the art. The CDRs are
parts of immunoglobulins that determine the specificity of said
molecules and make contact with a specific ligand. The CDRs are the
most variable part of the molecule and contribute to the diversity
of these molecules. There are three CDR regions CDR1, CDR2 and CDR3
in each V domain. CDR-H depicts a CDR region of a variable heavy
chain and CDR-L relates to a CDR region of a variable light chain.
VH means the variable heavy chain and VL means the variable light
chain. The CDR regions of an Ig-derived region may be determined as
described in Kabat "Sequences of Proteins of Immunological
Interest", 5th edit. NIH Publication no. 91-3242 U.S. Department of
Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987),
901-917 or Chothia Nature 342 (1989), 877-883.
Accordingly, in the context of the present invention, the antibody
molecule described herein above is selected from the group
consisting of a full antibody (immunoglobulin, like an IgG1, an
IgG2, an IgG2a, an IgG2b, an IgA1, an IgGA2, an IgG3, an IgG4, an
IgA, an IgM, an IgD or an IgE), F(ab)-, Fab'-SH-, Fv-, Fab'-,
F(ab')2-fragment, a chimeric antibody, a CDR-grafted antibody, a
fully human antibody, a bivalent antibody-construct, an
antibody-fusion protein, a synthetic antibody, bivalent single
chain antibody, a trivalent single chain antibody and a multivalent
single chain antibody.
"Humanization approaches" are well known in the art and in
particular described for antibody molecules, e.g. Ig-derived
molecules. The term "humanized" refers to humanized forms of
non-human (e.g., murine) antibodies or fragments thereof (such as
Fv, Fab, Fab', F(ab'), scFvs, or other antigen-binding partial
sequences of antibodies) which contain some portion of the sequence
derived from non-human antibody. Humanized antibodies include human
immunoglobulins in which residues from a complementary determining
region (CDR) of the human immunoglobulin are replaced by residues
from a CDR of a non-human species such as mouse, rat or rabbit
having the desired binding specificity, affinity and capacity. In
general, the humanized antibody will comprise substantially all of
at least one, and generally two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin; see, inter alia, Jones et al., Nature 321
(1986), 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596.
Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acids
introduced into it from a source which is non-human still retain
the original binding activity of the antibody. Methods for
humanization of antibodies/antibody molecules are further detailed
in Jones et al., Nature 321 (1986), 522-525; Reichmann et al.,
Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239
(1988), 1534-1536. Specific examples of humanized antibodies, e.g.
antibodies directed against EpCAM, are known in the art, see e.g.
(LoBuglio, Proceedings of the American Society of Clinical Oncology
Abstract (1997), 1562 and Khor, Proceedings of the American Society
of Clinical Oncology Abstract (1997), 847).
Accordingly, in the context of this invention, antibody molecules
or antigen-binding fragments thereof are provided, which are
humanized and can successfully be employed in pharmaceutical
compositions.
Moreover, in a preferred embodiment, the antibody of the present
invention is an antibody or antigen-binding fragment thereof that
binds to the glycoprotein B (gB) of HSV-1 and/or HSV-2 which
comprises or consists of VH domain (heavy chain variable region)
and VL domain (light chain variable region), i.e., the amino acid
sequence of the variable region of the heavy chain of an antibody
as depicted in SEQ ID NO:9 and the amino acid sequence of the
variable region of the light chain of an antibody as depicted in
SEQ ID NO:10.
However, the antibody or antigen-binding fragment thereof as used
in the present invention is not particularly limited to such
variable heavy and light chain variable regions but may also be an
antibody or antigen-binding fragment thereof that binds to the
glycoprotein B (gB) of HSV-1 and/or HSV-2 envelope which comprises
or consists of VH domain and VL domain with at least 95%, 90%, 85%,
75%, 70%, 65%, 60%, 55% or 50% sequence homology with the sequences
of SEQ ID NOs: 9 and 10, respectively, as long as the antibody or
antigen-binding fragment has the capability of having an effect in
treating an acute infection of mucosal or epidermal tissue in a
subject caused by HSV-1 or HSV-2 in terms of the present invention
or being capable of inhibiting the spreading of HSV from an
infected cell to an adjacent second non-infected cell (cell-to-cell
spread) or being capable of inhibiting cell-to-cell spread
independent from antibody-dependent cellular cytotoxicity (ADCC)
and/or complement-dependent cytotoxicity (CDC) as described herein
above and below. Furthermore, the antibody or antigen-binding
fragment thereof is a molecule that comprises VH and VL domains
having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative
amino acid substitutions with reference to the sequences of SEQ ID
NOs: 9 and 10. Moreover, the antibody or antigen-binding fragment
thereof is an antibody fragment selected from the group consisting
of Fab, Fab', Fab'-SH, FV, scFV, F(ab')2, and a diabody.
In order to determine whether an amino acid sequence has a certain
degree of identity to the sequences of SEQ ID NOs: 9 and 10, the
skilled person can use means and methods well known in the art,
e.g. alignments, either manually or by using computer programs
known to the person skilled in the art. Such an alignment can,
e.g., be done with means and methods known to the skilled person,
e.g. by using a known computer algorithm such as the Lipman-Pearson
method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is
preferred that in such an alignment maximum homology is assigned to
conserved amino acid residues present in the amino acid sequences.
In a preferred embodiment ClustalW2 is used for the comparison of
amino acid sequences. In the case of pairwise
comparisons/alignments, the following settings are preferably
chosen: Protein weight matrix: BLOSUM 62; gap open: 10; gap
extension: 0.1. In the case of multiple comparisons/alignments, the
following settings are preferably chosen: Protein weight matrix:
BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no
end gap.
In accordance with the present invention, the term "identical" or
"percent identity" in the context of two or more nucleic acid or
amino acid sequences, refers to two or more sequences or
subsequences that are the same, or that have a specified percentage
of amino acid residues or nucleotides that are the same (e.g., 60%
or 65% identity, preferably, 70-95% identity, more preferably at
least 95% identity with the nucleic acid sequences or with the
amino acid sequences as described above which are capable of
binding to gB of HSV-1 or HSV-2 and having the capability of
treating an acute infection of mucosal or epidermal tissue in a
subject caused by HSV-1 or HSV-2 in terms of the present invention
or being capable of inhibiting the spreading of HSV from an
infected cell to an adjacent second non-infected cell (cell-to-cell
spread) or being capable of inhibiting cell-to-cell spread
independent from antibody-dependent cellular cytotoxicity (ADCC)
and/or complement-dependent cytotoxicity (CDC) as described herein
above and below), when compared and aligned for maximum
correspondence over a window of comparison, or over a designated
region as measured using a sequence comparison algorithm as known
in the art, or by manual alignment and visual inspection. Sequences
having, for example, 60% to 95% or greater sequence identity are
considered to be substantially identical. Such a definition also
applies to the complement of a test sequence. Preferably, the
described identity exists over a region that is at least about 15
to 25 amino acids or nucleotides in length, more preferably, over a
region that is about 50 to 100 amino acids or nucleotides in
length. Those having skill in the art will know how to determine
percent identity between/among sequences using, for example,
algorithms such as those based on CLUSTALW computer program
(Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag
Comp. App. Biosci. 6 (1990), 237-245), as known in the art.
Although the FASTDB algorithm typically does not consider internal
non-matching deletions or additions in sequences, i.e., gaps, in
its calculation, this can be corrected manually to avoid an
overestimation of the % identity. CLUSTALW, however, does take
sequence gaps into account in its identity calculations. Also
available to those having skill in this art are the BLAST and BLAST
2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402;
Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol.
Biol. 215:403-410). The BLASTN program for nucleic acid sequences
uses as defaults a word length (W) of 11, an expectation (E) of 10,
M=5, N=4, and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength (W) of
3, and an expectation (E) of 10. The BLOSUM62 scoring matrix
(Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50,
expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
Preferably, the amino acid substitution(s) are "conservative
substitution(s)" which refers to substitutions of amino acids in a
protein with other amino acids having similar characteristics (e.g.
charge, side-chain size, hydrophobicity/hydrophilicity, backbone
conformation and rigidity, etc.), such that the changes can
frequently be made without altering the biological activity of the
protein. Those of skill in this art recognize that, in general,
single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see,
e.g., Watson Molecular Biology of the Gene, The Benjamin/Cummings
Pub. Co. 4th Ed. (1987), 224. In addition, substitutions of
structurally or functionally similar amino acids are less likely to
disrupt biological activity. Within the context of the present
invention the binding compounds/antibodies of the present invention
comprise polypeptide chains with sequences that include up to 0 (no
changes), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more
conservative amino acid substitutions when compared with the
specific amino acid sequences disclosed herein, for example, SEQ ID
NO: 9 (referring to the variable region of the antibody heavy chain
of the antibody) and 10 (referring to the variable of the light
chain of the antibody). As used herein, the phrase "up to X"
conservative amino acid substitutions includes 0 substitutions and
any number of substitutions up to 10 and including 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 substitutions.
Such exemplary substitutions are preferably made in accordance with
those set forth in Table 1 as follows:
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Original Conservative residue substitution Ala (A)
Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
Moreover, in a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention comprises an amino acid sequence with at least 70%
sequence identity to the amino acid residues shown in positions 1
to 30, 38 to 51, 68 to 99, and 112 to 122 of SEQ ID NO: 7 and in
positions 1 to 23, 41 to 55, 63 to 94, and 104 to 114 of SEQ ID NO:
8.
In a further, preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention comprises an amino acid sequence with at least 75%, at
least 80%, more preferably at least 85%, at least 90%, even more
preferably at least 95%, and most preferably 98% overall sequence
identity in the framework regions compared to the amino acid
residues shown in positions 1 to 30, 38 to 51, 68 to 99, and 112 to
122 of SEQ ID NO: 7 and in positions 1 to 23, 41 to 55, 63 to 94,
and 104 to 114 of SEQ ID NO: 8. Such antibodies are suitable for
the medical uses of the present invention as long as the antibody
or antigen-binding fragment binds to gB of HSV-1 or HSV-2 and has
the capability of having an effect in treating an acute infection
of mucosal or epidermal tissue in a subject caused by HSV-1 or
HSV-2 in terms of the present invention or being capable of
inhibiting the spreading of HSV from an infected cell to an
adjacent second non-infected cell (cell-to-cell spread) or being
capable of inhibiting cell-to-cell spread independent from
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC) as described herein above
and below.
Thus, in a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention comprises an amino acid sequence having the above
variable regions of the light and heavy chains (i.e., the CDRs
defined above, i.e., V.sub.HCDR1 comprising SEQ ID NO: 1,
V.sub.HCDR2 comprising SEQ ID NO: 2, V.sub.HCDR3 comprising SEQ ID
NO: 3, V.sub.LCDR1 comprising SEQ ID NO: 4, V.sub.LCDR2 comprising
SEQ ID NO: 5, and V.sub.LCDR3 comprising SEQ ID NO:6) while the
amino acid sequence have a variability in the framework region with
at least 75%, at least 80%, more preferably at least 85%, at least
90%, even more preferably at least 95%, and most preferably 98%
overall sequence identity in the framework regions compared to the
amino acid residues shown in positions 1 to 30, 38 to 51, 68 to 99,
and 112 to 122 of SEQ ID NO: 7 and in positions 1 to 23, 41 to 55,
63 to 94, and 104 to 114 of SEQ ID NO: 8.
In this context, a polypeptide has "at least X % sequence identity"
in the framework regions to SEQ ID NO:7 or 8 if SEQ ID NO:7 or SEQ
ID NO: 8 is aligned with the best matching sequence of a
polypeptide of interest and the amino acid identity between those
two aligned sequences is at least X % over positions 1 to 30, 38 to
51, 68 to 99, and 112 to 122 of SEQ ID NO: 7 and positions 1 to 23,
41 to 55, 63 to 94, and 104 to 114 of SEQ ID NO: 8. As mentioned
above, such an alignment of amino acid sequences can be performed
using, for example, publicly available computer homology programs
such as the "BLAST" program provided on the National Centre for
Biotechnology Information (NCBI) homepage using default settings
provided therein. Further methods of calculating sequence identity
percentages of sets of amino acid sequences or nucleic acid
sequences are known in the art.
Moreover, in a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention comprises the V.sub.H of SEQ ID NO:9 and the V.sub.L of
SEQ ID NO:10.
The specificity of the antibody or antigen-binding fragment of the
present invention may not only be expressed by the nature of the
amino acid sequence of the antibody or the antigen-binding fragment
as defined above but also by the epitope to which the antibody is
capable of binding to. Thus, the present invention utilizes in a
preferred embodiment an anti-HSV antibody or an antigen-binding
fragment thereof for use according to the present invention which
recognizes the same epitope as the antibody as described above,
preferably the mAbhu2c. As shown in the Examples section and as
illustrated in FIGS. 13A and 13B of WO2011/038933 A2, this epitope
is a discontinuous or rather a pseudocontinuous epitope partially
resistant to denaturation located at the amino acids 172-195 and
295-313 of glycoprotein B of HSV-1 and HSV-2. In the context of the
present application, the epitope of the mAb 2c antibody may be
located within the first 487 amino-terminal residues of the gB
protein. Preferably, the epitope may comprise at least one amino
acid sequence located within the amino acid sequence between
position 172 and 307 of the gB protein.
The epitope may comprise the consecutive amino acid sequence
.sub.301YGYRE.sub.305 of the gB protein, preferably the consecutive
amino acid sequence .sub.301YGYREG.sub.306 or
.sub.300FYGYRE.sub.305, more preferably the sequence may be further
extended at the termini (i.e., .sub.299PFYGYRE.sub.305 or
.sub.300FYGYREGS.sub.307). The epitope of the antibodies of the
present invention may comprise the consecutive amino acid sequence
298-313 (.sub.298SPFYGYREGSHTEHTS.sub.313) of gB.
Alternatively, the epitope may be located in the consecutive amino
acid sequence .sub.172QVWFGHRYSQFMGIFED.sub.188. The epitope may
comprise the consecutive amino acid sequence
.sub.172QVWFGHRYSQFMG.sub.184.
Preferably, the epitope may be consisted of more than one
consecutive amino acid sequences. The epitope may partly be a
discontinuous epitope. More preferably, the epitope may comprise
two consecutive amino acid sequences. Such an epitope consisting of
two amino acid sequences may be designated as "duotope". The
antibody may bind to both amino acid sequences.
More preferably, the amino acid sequences of the duotope may
comprise the amino acid sequence .sub.300FYGYRE.sub.305 and an
amino acid sequence located between amino acid position 172 and
188. Even more preferably, the epitope may comprise the amino acid
sequence .sub.300FYGYRE.sub.305 and amino acid sequence
.sub.179YSQFMG.sub.184 of the gB protein. Alternatively, the
epitope or the duotope may be chemically synthesized. The epitope
may be a chemically synthesized epitope having the sequence
YSQFMG-.beta.A-FYGYRE. The abbreviation .beta.A as used herein
refers to beta-alanine.
Most preferably, the epitope may comprise the amino acid sequence
FYGYRE and amino acid sequence FED of the gB protein. The epitope
may be a chemically synthesized epitope having the sequence
FED-.beta.A-.beta.A-FYGYRE or PFYGYREGFEDF.
It may be understood by a person skilled in the art that the
epitopes may be comprised in the gB protein, but may also be
comprised in a degradation product thereof or may be a chemically
synthesized peptide. The amino acid positions are only indicated to
demonstrate the position of the corresponding amino acid sequence
in the sequence of the gB protein. The invention encompasses all
peptides comprising the epitope. The peptide may be a part of a
polypetide of more than 100 amino acids in length or may be a small
peptide of less than 100, preferably less than 50, more preferably
less than 25 amino acids, even more preferably less than 16 amino
acids. The amino acids of such peptide may be natural amino acids
or nonnatural amino acids (e.g., beta-amino acids, gamma-amino
acids, D-amino acids) or a combination thereof. Further, the
present invention may encompass the respective retro-inverso
peptides of the epitopes. The peptide may be unbound or bound. It
may be bound, e.g., to a small molecule (e.g., a drug or a
fluorophor), to a high-molecular weight polymer (e.g., polyethylene
glycol (PEG), polyethylene imine (PEI), hydroxypropylmethacrylate
(HPMA), etc.) or to a protein, a fatty acid, a sugar moiety or may
be inserted in a membrane.
In order to test whether an antibody in question and the antibody
of the present invention recognize the same epitope, the following
competition study may be carried out: Vero cells infected with 3
moi (multiplicity of infection) are incubated after 20 h with
varying concentrations of the antibody in question as the
competitor for 1 hour. In a second incubation step, the antibody of
the present invention is applied in a constant concentration of 100
nM and its binding is flow-cytometrically detected using a
fluorescence-labelled antibody directed against the constant
domains of the antibody of the invention. Binding that conducts
anti-proportional to the concentration of the antibody in question
is indicative for that both antibodies recognize the same epitope.
However, many other assays are known in the art which may be
used.
Thus, in a preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use according to the present
invention recognizes the same epitope as mAb 2c, wherein said
epitope is located at the amino acids 172-195 and 295-315 of
glycoprotein B of HSV-1 and HSV-2. Using overlapping 15-mer
peptides spanning the gB region from amino acid 31 to 505 it has
been described in Daumer et al., Med Microbiol Immunol 2011
(200):85-97 that the mAb 2c is capable of recognizing an epitope
which is located at the amino acids 175-195 and 298-315 of
glycoprotein B of HSV-1 and HSV-2. Using high-resolution 13-mer
peptide microarrays Krawczyk et al., Journal of Virology 2011
(85):1793-1803 mapped the epitope recognized by mAb 2c to the amino
acids 172-195 and 295-313 of glycoprotein B of HSV-1 and HSV-2.
The sequence of the glycoprotein B of HSV-1 and/or HSV-2 is
well-characterized and, as defined above, without being bound to
specific sequences, examples sequences of various HSV-1 and HSV-2
strains, respectively, are shown in SEQ ID NOs:11 to 16. The
epitope recognized by the mAb 2c antibody is highly conserved among
various HSV-strains as well as between HSV-1 and HSV-2.
This antibody or the antigen-binding fragment thereof which may be
used in the treatment as disclosed in the present invention is not
limited to the antibody detecting the above epitope of glycoprotein
B of HSV-1 and HSV-2. In fact, also other antibodies which detect
another epitope of glycoprotein B or even an epitope of another
protein or polypeptide of HSV-1 and HSV-2 can be used in the
treatment of the present invention as long as such an antibody is
capable of having an effect in treating an acute infection of
mucosal or epidermal tissue in a subject caused by HSV-1 or HSV-2
in terms of the present invention or being capable of inhibiting
the spreading of HSV from an infected cell to an adjacent second
non-infected cell (cell-to-cell spread) or being capable of
inhibiting cell-to-cell spread independent from antibody-dependent
cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC) as described herein above and below.
With the normal skill of the person skilled in the art and by
routine methods, the person skilled in the art can easily deduce
from the sequences provided herein relevant epitopes (also
functional fragments) of the polypeptides of HSV which are useful
in the generation of antibodies like polyclonal and monoclonal
antibodies. However, the person skilled in the art is readily in a
position to also provide for engineered antibodies like CDR-grafted
antibodies or also humanized and fully human antibodies and the
like.
Particularly preferred in the context of the present invention are
monoclonal antibodies. For the preparation of monoclonal
antibodies, any technique which provides antibodies produced by
continuous cell line cultures can be used. Examples for such
techniques include the hybridoma technique, the trioma technique,
the human B-cell hybridoma technique and the EBV-hybridoma
technique to produce human monoclonal antibodies (Shepherd and Dean
(2000), Monoclonal Antibodies: A Practical Approach, Oxford
University Press, Goding and Goding (1996), Monoclonal Antibodies:
Principles and Practice--Production and Application of Monoclonal
Antibodies in Cell Biology, Biochemistry and Immunology, Academic
Pr Inc, USA).
The antibody derivatives can also be produced by peptidomimetics.
Further, techniques described for the production of single chain
antibodies (see, inter alia, U.S. Pat. No. 4,946,778) can be
adapted to produce single chain antibodies specifically recognizing
an antigen of HSV. Also, transgenic animals may be used to express
humanized antibodies to the polypeptide of HSV.
The present invention also envisages the production of specific
antibodies against native polypeptides and recombinant polypeptides
of glycoprotein B or any another protein or polypeptide of HSV-1
and HSV-2. This production is based, for example, on the
immunization of animals, like mice. However, also other animals for
the production of antibody/antisera are envisaged within the
present invention. For example, monoclonal and polyclonal
antibodies can be produced by rabbit, mice, goats, donkeys and the
like. The polynucleotide encoding a correspondingly chosen
polypeptide of HSV-1 or HSV-2 can be subcloned into an appropriated
vector, wherein the recombinant polypeptide is to be expressed in
an organism being able for an expression, for example in bacteria.
Thus, the expressed recombinant protein can be intra-peritoneally
injected into a mice and the resulting specific antibody can be,
for example, obtained from the mice serum being provided by
intra-cardiac blood puncture. The present invention also envisages
the production of specific antibodies against native polypeptides
and recombinant polypeptides by using a DNA vaccine strategy as
exemplified in the appended examples. DNA vaccine strategies are
well-known in the art and encompass liposome-mediated delivery, by
gene gun or jet injection and intramuscular or intradermal
injection. Thus, antibodies directed against a polypeptide or a
protein or an epitope of HSV-1 and HSV-2 can be obtained by
directly immunizing the animal by directly injecting
intramuscularly the vector expressing the desired polypeptide or a
protein or an epitope of HSV-1 and HSV-2, in particular an epitope
of gB. The amount of obtained specific antibody can be quantified
using an ELISA, which is also described herein below. Further
methods for the production of antibodies are well known in the art,
see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH
Press, Cold Spring Harbor, 1988. The term "specifically binds", as
used herein, refers to a binding reaction that is determinative of
the presence of the desired polypeptide or a protein or an epitope
of HSV-1 and HSV-2, in particular an epitope of gB, and an antibody
in the presence of a heterogeneous population of proteins and other
biologics.
Thus, under designated assay conditions, the specified antibodies
and a corresponding polypeptide or a protein or an epitope of HSV-1
and HSV-2, in particular an epitope of gB, bind to one another and
do not bind in a significant amount to other components present in
a sample. Specific binding to a target analyte under such
conditions may require a binding moiety that is selected for its
specificity for a particular target analyte. A variety of
immunoassay formats may be used to select antibodies specifically
reactive with a particular antigen. For example, solid-phase ELISA
immunoassays are routinely used to select monoclonal antibodies
specifically immunoreactive with an analyte. See Shepherd and Dean
(2000), Monoclonal Antibodies: A Practical Approach, Oxford
University Press and/or Howard and Bethell (2000) Basic Methods in
Antibody Production and Characterization, Crc. Pr. Inc. for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity. Typically a specific or
selective reaction will be at least twice background signal to
noise and more typically more than 10 to 100 times greater than
background. The person skilled in the art is in a position to
provide for and generate specific binding molecules directed
against the novel polypeptides. For specific binding-assays it can
be readily employed to avoid undesired cross-reactivity, for
example polyclonal antibodies can easily be purified and selected
by known methods (see Shepherd and Dean, loc. cit.).
The term "anti-HSV antibody or antigen-binding fragment thereof"
means in accordance with this invention that the antibody molecule
or antigen-binding fragment thereof is capable of specifically
recognizing or specifically interacting with and/or binding to at
least two amino acids of the desired polypeptide or a protein or an
epitope of HSV-1 and HSV-2, in particular an epitope of gB. Said
term relates to the specificity of the antibody molecule, i.e. to
its ability to discriminate between the specific regions a desired
polypeptide or a protein or an epitope of HSV-1 and HSV-2, in
particular an epitope of gB. Accordingly, specificity can be
determined experimentally by methods known in the art and methods
as disclosed and described herein. Such methods comprise, but are
not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and
peptide scans. Such methods also comprise the determination of
K.sub.D-values as, inter alia, illustrated in the appended
examples. The peptide scan (pepspot assay) is used routinely
employed to map linear epitopes in a polypeptide antigen. The
primary sequence of the polypeptide is synthesized successively on
activated cellulose with peptides overlapping one another. The
recognition of certain peptides by the antibody to be tested for
its ability to detect or recognize a specific antigen/epitope is
scored by routine colour development (secondary antibody with
horseradish peroxide and 4-chloronaphtol and hydrogenperoxide), by
a chemoluminescence reaction or similar means known in the art. In
the case of, inter alia, chemoluminescence reactions, the reaction
can be quantified. If the antibody reacts with a certain set of
overlapping peptides one can deduce the minimum sequence of amino
acids that are necessary for reaction. The same assay can reveal
two distant clusters of reactive peptides, which indicate the
recognition of a discontinuous, i.e. conformational epitope in the
antigenic polypeptide (Geysen (1986), Mol. Immunol. 23,
709-715).
A preferred epitope of the anti-HSV antibody or antigen-binding
fragment thereof is defined above and below is the same that is
recognized by the mAb2c.
In a preferred embodiment, the anti-HSV antibody (or an
antigen-binding fragment thereof) for use according to the present
invention is the mAb 2c antibody (or an antigen-binding fragment
thereof). This monoclonal antibody MAb 2c has been described
elsewhere and has been demonstrated to neutralize virus by
abrogating viral cell-to-cell spread, a key mechanism by which
HSV-1/2 escapes humoral immune surveillance independent from
antibody-dependent cellular cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC); Eis-Hubinger et al.,
Intervirology 32:351-360 (1991); Eis-Hubinger et al., Journal of
General Virology 74:379-385 (1993); WO2011/038933 A2; Krawczyk A,
et al., Journal of virology (2011); 85(4):1793-1803; Krawczyk A, et
al., Proc Natl Acad Sci USA (2013); 110(17):6760-6765.
The antibodies and antigen-binding-fragments thereof as defined
above are particularly useful in medical settings involving the
topical administration. Thus, as mentioned above, the present
invention relates to the medical use of an anti-HSV antibody or
antigen-binding fragment thereof wherein said antibody or
antigen-binding fragment thereof is topically administered.
Accordingly, the present invention relates to an anti-HSV antibody
or an antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum, wherein said antibody is to be topically
administered.
The term "treatment" and the like are used herein to generally mean
obtaining a desired pharmacological and/or physiological effect. As
already described above, the treatment of the present invention
relates to the treatment of acute infections and, accordingly,
excludes that the effect may be prophylactic in terms of completely
or partially preventing a disease or symptom thereof. Rather, the
term "treatment" is to be understood as being therapeutic in terms
of partially or completely curing a disease and/or adverse effect
and/or symptoms attributed to the disease of an acute HSV infection
as defined above. Hence, the treatment of the present invention
relates to the treatment of acute infections. "Acute" in this
respect means that the subject shows symptoms of the disease. In
other words, the subject to be treated is in actual need of a
treatment and the term "acute treatment" in the context of the
present invention relates to the measures taken to actually treat
the disease after the onset of the disease or the breakout of the
disease. The term "acute" as referred to in the context of the
present invention is opposed to a prophylactic treatment or
preventive treatment, i.e., measures taken for disease prevention,
e.g., in order to prevent the infection and/or the onset of the
disease. More specifically, prophylactic treatment may be
understood in a way that it prevents attachment of free virus
particles (from outside the body) to target cells and in turn
prevents virus replication. In contrast, at an acute infection
(which could be a primary or a recurrent infection) progeny virus
have been raced upon HSV replication. Thus, the "acute treatment"
referred to in the present invention does explicitly not relate to
prophylactic or preventive treatment of an infection caused by
HSV-1 or HSV-2.
Topical administration in accordance with the present invention
relates to a medication or application or administration that is
applied to body surfaces such as the skin or mucous membranes to
treat the infection referred to above via a large range of classes
of forms of administration, including but not limited to creams,
foams, gels, lotions and ointments. In a preferred embodiment,
topical administration is understood to be epicutaneous, meaning
that the anti-HSV antibody or an antigen-binding fragment thereof
is applied directly to the skin. Without being bound by theory and
to provide some further non-limiting examples, topical application
may also be inhalational, such as asthma medications, or applied to
the surface of tissues other than the skin, such as eye drops
applied to the conjunctiva, or ear drops placed in the ear, or
medications applied to the surface of a tooth. As a route of
administration, topical administration are contrasted with enteral
(in the digestive tract) and intravascular/intravenous (injected
into the circulatory system). In its broadest sense, a topical
effect may be understood in a way that it relates to, in the
pharmacodynamic sense, a local, rather than systemic, target for a
medication.
The mode of topical administration in accordance with the present
invention, i.e., the medication, pharmaceutical composition or
application or administration that is applied to body surfaces such
as the skin or mucous membranes to treat the infection of acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum is not particularly limited and the skilled person knows
many forms and preparations that may be suitable for topical
administration. Without being bound by theory and without being
limiting, the following examples are given. There are many general
classes, with no clear dividing line between similar formulations
suitable for topical medication. As an example, a topical solution
may be used. Topical solutions are generally of low viscosity and
often use water or alcohol in the base.
As another example, a lotion may be used to administer the anti-HSV
antibody topically. Lotions are similar to solutions but are
thicker and tend to be more emollient in nature than solution. They
are usually an oil mixed with water, and more often than not have
less alcohol than solutions.
As another example, a cream may be used to administer the anti-HSV
antibody topically. A cream is usually an emulsion of oil and water
in approximately equal proportions. It penetrates the stratum
corneum outer layer of skin well. Cream is thicker than lotion, and
maintains its shape when removed from its container. It tends to be
moderate in moisturizing tendency.
As another example, an ointment may be used to administer the
anti-HSV antibody topically. An ointment is commonly a homogeneous,
viscous, semi-solid preparation, most commonly a greasy, thick oil
(oil 80%-water 20%) with a high viscosity, that is intended for
external application to the skin or mucous membranes. Ointments
have a Water number that defines the maximum amount of water that
it can contain. They may be used as emollients or for the
application of the anti-HSV antibody in accordance with the present
invention to the skin for protective, therapeutic, or prophylactic
purposes and where a degree of occlusion is desired. The vehicle of
an ointment is known as the ointment base. The choice of a base
depends upon the clinical indication for the ointment and is
appropriately chosen based on the person skilled in the art's
knowledge. Different types of ointment bases may be hydrocarbon
bases, e.g. hard paraffin, soft paraffin, microcrystalline wax and
ceresine; absorption bases, e.g. wool fat, beeswax; water soluble
bases, e.g. macrogols 200, 300, 400; emulsifying bases, e.g.
emulsifying wax, cetrimide; vegetable oils, e.g. olive oil, coconut
oil, sesame oil, almond oil and peanut oil. Commonly, the
medicament, i.e., the anti-HSV antibody in the present invention,
is dispersed in the base, and later they get divided after the drug
penetration into the living cells of skin. Ointments are commonly
formulated using hydrophobic, hydrophilic, or water-emulsifying
bases to provide preparations that are immiscible, miscible, or
emulsifiable with skin secretions. They can also be derived from
hydrocarbon (fatty), absorption, water-removable, or water-soluble
bases.
As another example, a gel may be used to administer the anti-HSV
antibody topically. Gels are usually thicker than a solution. Gels
are often a semisolid emulsion in an alcohol base. Some will melt
at body temperature. Gel tends to be cellulose cut with alcohol or
acetone.
As another example, a foam may be used to administer the anti-HSV
antibody topically.
As another example, a transdermal patch may be used to administer
the anti-HSV antibody topically. Transdermal patches can be a very
precise time released method of delivering a drug. The release of
the active component from a transdermal delivery system (patch) may
be controlled by diffusion through the adhesive which covers the
whole patch, by diffusion through a membrane which may only have
adhesive on the patch rim or drug release may be controlled by
release from a polymer matrix. As another example, a powder may be
used to administer the anti-HSV antibody topically. Powder is
either the pure drug by itself (talcum powder), or is made of the
drug mixed in a carrier such as corn starch or corn cob powder
(Zeosorb AF--miconazole powder).
As another example, a solid form may be used to administer the
anti-HSV antibody topically. Thus, the anti-HSV antibody may be
placed in a solid form. Examples are deodorant, antiperspirants,
astringents, and hemostatic agents. In a preferred embodiment, in
particular in the context of the topical administration of the
anti-HSV antibody in the treatment of an acute infection of mucosal
or epidermal tissue caused by HSV-1 or HSV-2 of Herpes simplex
genitalis, the anti-HSV antibody may be administered in the form of
a suppository. A suppository is a drug delivery system that in the
context of the treatment of Herpes simplex genitalis comprises the
anti-HSV antibody and may be is inserted into the vagina (i.e., in
the form of a vaginal suppository), where it dissolves or melts and
releases the anti-HSV antibody and, accordingly serves to deliver
locally the anti-HSV-antibody.
As another example, a vaporizing device may be used to administer
the anti-HSV antibody topically. Thus, the anti-HSV antibody may be
applied as an ointment or gel, and reach the mucous membrane via
vaporization.
As another example, a paste may be used to administer the anti-HSV
antibody topically. Paste combines three agents--oil, water, and
powder. It is an ointment in which a powder is suspended.
As a final, non-limiting example, a tincture may be used to
administer the anti-HSV antibody topically. A tincture is a skin
preparation that has a high percentage of alcohol.
In another embodiment, the anti-HSV antibody or an antigen-binding
fragment thereof for use in treating an acute infection of mucosal
or epidermal tissue in a subject caused by HSV-1 or HSV-2 selected
from the group consisting of Herpes simplex labialis, Herpes
simplex genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum, is to be
topically applied to infected mucosal or epidermal tissue. The area
the anti-HSV antibody or an antigen-binding fragment thereof is to
be applied to is not particularly limited. Preferably, an area of
the mucosal or epidermal tissue is chosen which displays acute
symptoms of an infection caused by HSV-1 or HSV-2. Preferably,
these areas or parts of the subject's body are the lips, genitals,
nose, ears, eyes, fingers, toes and/or skin areas throughout the
body, preferably on the head, the jaw area, neck, chest, face,
stomach and/or legs. In particular, in cutaneaous Herpes simplex
infection as described above, commonly (larger) skin areas
throughout the body can be affected while in Herpes gladiatorum as
described above, it usually occurs on the head, most commonly the
jaw area, the neck, chest, face, stomach, and legs. Accordingly, in
these diseases, it is preferred that the topical administration in
accordance with the invention is effected to these body parts or
areas of mucosal or epidermal tissue.
In a further preferred embodiment, the anti-HSV antibody or the
antigen-binding fragment thereof for use in accordance with the
present invention is to be topically applied to areas surrounding
the infected mucosal or epidermal tissue. Areas surrounding the
infected mucosal or epidermal tissue are to be understood as the
area around a given infected location of the tissue. The extent of
the area of the surrounding is not particularly limiting but may
cover, e.g., an area adjacent to/surrounding the infected tissue
which has approximately 0.5 times the size of the infected area,
the same size of the infected area, 1.5 times, preferably 2 times
or even more preferably 3, 4 or 5 times the size of the infected
area.
The anti-HSV antibody or an antigen-binding fragment thereof for
use in treating an acute infection of mucosal or epidermal tissue
in a subject caused by HSV-1 or HSV-2 selected from the group
consisting of Herpes simplex labialis, Herpes simplex genitalis,
chronic or disseminated cutaneous herpes simplex infection, Herpes
gladiatorum and Eczema herpeticum, wherein said anti-HSV antibody
or an antigen-binding fragment thereof is to be topically applied
may be administered in combination with a virostatic agent.
Preferably, such a combination therapy exerts synergistic effects
on the treatment in accordance with the present invention.
The term "combination" as used herein relates to a combination of
anti-HSV antibody or an antigen-binding fragment thereof as
outlined above and a virostatic agent described herein below. In a
preferred embodiment, a simultaneous application is envisaged. Yet,
the combination also encompasses a subsequent application of the
two components, i.e. anti-HSV antibody or an antigen-binding
fragment thereof as outlined above and a virostatic agent described
herein below. Thus, one of these components may be administered
before, simultaneously with or after the other one of the
combination, or vice versa. Accordingly, "in combination" as used
herein does not restrict the timing between the administration of
the anti-HSV antibody or an antigen-binding fragment thereof as
outlined above and a virostatic agent described herein below. Thus,
when the two components are not administered simultaneously
with/concurrently, the administrations may be separated by 1
minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours
or by any suitable time differential readily determined by one of
skill in art and/or described herein.
Virostatic agents are well-known to the person skilled in the art
and are commonly also referred to as antiviral drugs which are a
class of medication used specifically for treating viral
infections. Specific antivirals are used for specific viruses.
Unlike most antibiotics, antiviral drugs do not destroy their
target pathogen; instead they inhibit their development.
With respect to HSV infections, the skilled person is in a position
to select an appropriate virostatic agent that is suitable to
inhibit the virus' development in accordance with the present
invention. As examples, virostatic agent may be selected from the
group consisting of the drug classes of nucleoside analogues,
pyrophosphate analogues, nucleotide analogues, amantadin
derivatives and helicase-primase inhibitors. Thus, the present
invention relates to an anti-HSV antibody or an antigen-binding
fragment thereof for use in treating an acute infection of mucosal
or epidermal tissue in a subject caused by HSV-1 or HSV-2 selected
from the group consisting of Herpes simplex labialis, Herpes
simplex genitalis, chronic or disseminated cutaneous herpes simplex
infection, Herpes gladiatorum and Eczema herpeticum, wherein said
anti-HSV antibody or an antigen-binding fragment thereof is to be
topically applied in combination with a virostatic agent selected
from the group consisting of the drug classes of nucleoside
analogues, pyrophosphate analogues, nucleotide analogues, and
helicase-primase inhibitors.
Nucleoside analogues are known in the art and relate to molecules
that act like nucleosides in DNA synthesis. They include a range of
antiviral products used to prevent viral replication in infected
cells. Once they are phosphorylated, they work as antimetabolites
by being similar enough to nucleotides to be incorporated into
growing DNA strands, but they act as chain terminators and stop
viral DNA Polymerase. Nucleoside, nucleotide and pyrophosphate
analogues in general are known to inhibit viral nucleic acid
synthesis to block viral replication. Nucleoside, nucleotide
analogues are antimetabolite drugs. Pyrophosphate analogues (e.g.
Foscarnet) structurally mimic the anion pyrophosphate and exert
antiviral activity by a selective inhibition of the pyrophosphate
binding site on virus-specific DNA polymerases at concentrations
that do not affect cellular DNA polymerases. Nucleotide and
pyrophosphate analogues do not require an initial activation
(phosphorylation) by thymidine kinases or other kinases before
taken up into cells. Helicase-primase inhibitors are
non-nucleosidic inhibitors that target the viral
helicase-primase.
Preferably, commonly known and approved virostatic agents may be
used as summarized in the following. As a nucleoside analogue a
compound selected from the group consisting of Acyclovir,
Penciclovir, Valacyclovir and Famaciclovir may exemplarily be
mentioned and used in the combination therapy described above. As a
pyrophosphate analogue Foscarnet may be used. As a nucleotide
analogue Cidofovir may be used. As a helicase-primase inhibitor
Pritelivir is exemplarily mentioned. As an amantadine derivative,
Tromantandin may be used.
Acyclovir, also known as acycloguanosine (ACV) or
2-Amino-9-(2-hydroxyethoxymethyl)-3H-purin-6-on, is a guanosine
analogue antiviral drug, marketed under trade names such as,
ACERPES.RTM., Acic.RTM., Aciclobeta.RTM., AcicloCT.RTM.,
Aciclostad.RTM., Aciclovir, Acic.RTM., Ophtal.RTM., Acivir.RTM.,
AciVision, Acyclovir.RTM., Aviral.RTM., Cyclovir, Helvevir.RTM.,
Herpex, Supraviran.RTM., Virucalm.RTM., Virupos.RTM. Virzin,
Zoliparin.RTM., Zovir, and Zovirax.RTM..
Penciclovir
(2-amino-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6,9-dihydro-3H-purin-6-on)
is a guanine analogue antiviral drug, marketed under trade names
such as Denavir and Fenistil.
Famciclovir (2-[(acetyloxy)methyl]-4-(2-amino-9H-purin-9-yl)butyl
acetate) is a prodrug of penciclovir with improved oral
bioavailability.
Foscarnet is the conjugate base of the chemical compound with the
formula HO.sub.2CPO.sub.3H.sub.2 and is marketed under the trade
names Foscavir.RTM. and Triapten.RTM.. Valacyclovir, also known as
(S)-2-[(2-amino-6-oxo-6,9-dihydro-3H-purin-9-yl)methoxy]ethyl-2-amino-3-m-
ethylbutanoate, is a prodrug of the guanosine analogue antiviral
drug ACV marketed under the name e.g. Valtrex.RTM..
Cidovovir (CDV), also known as
(S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl)cytosine, is a
nucleotide analogue antiviral drug marketed under the name
Visitde.RTM..
Pritelevir is a thiazolylamide, also known as AIC-316, or BAY
57-1293, is a helicase-primase inhibitor currently in clinical
phase II trials for treatment of genital HSV-2 infections.
The local therapeutic drug Tromantandin (Viru-Merz Serol Gel) is
explicitly used for local treatment of HSV skin infections.
Tromantandin is an amantadin derivative. Griffin U.S. Pat. No.
4,351,847 discloses that an amantadine derivative is effective
against herpes simplex virus.
Moreover, the present invention relates to a pharmaceutical
composition, comprising an effective amount of the antibody or the
antigen-binding fragment thereof in accordance with the above and
at least one pharmaceutically acceptable excipient.
An excipient is an inactive substance formulated alongside the
active ingredient, i.e., the anti-HSV antibody or the
antigen-binding fragment thereof in accordance with the above, for
the purpose of bulking-up formulations that contain potent active
ingredients. Excipients are often referred to as "bulking agents,"
"fillers," or "diluents". Bulking up allows convenient and accurate
dispensation of a drug substance when producing a dosage form. They
also can serve various therapeutic-enhancing purposes, such as
facilitating drug absorption or solubility, or other
pharmacokinetic considerations. Excipients can also be useful in
the manufacturing process, to aid in the handling of the active
substance concerned such as by facilitating powder flowability or
non-stick properties, in addition to aiding in vitro stability such
as prevention of denaturation over the expected shelf life. The
selection of appropriate excipients also depends upon the route of
administration and the dosage form, as well as the active
ingredient and other factors.
Thus, in line with the above, the pharmaceutical composition
comprising an effective amount of the antibody or the
antigen-binding fragment thereof may be in solid, liquid or gaseous
form and may be, inter alia, in a form of (a) powder(s), (a)
tablet(s), (a) solution(s) or (an) aerosol(s). It is preferred that
said pharmaceutical composition optionally comprises a
pharmaceutically acceptable carrier and/or diluent.
Examples of suitable pharmaceutical carriers, excipients and/or
diluents are well known in the art and include phosphate buffered
saline solutions, water, emulsions, such as oil/water emulsions,
various types of wetting agents, sterile solutions etc.
Compositions comprising such carriers can be formulated by well
known conventional methods. These pharmaceutical compositions can
be administered to the subject at a suitable dose, i.e., in "an
effective amount" which can easily be determined by the skilled
person by methods known in the art. Administration of the suitable
pharmaceutical composition is effected in accordance with the
present invention by topical administration. The dosage regimen
will be determined by the attending physician and clinical factors.
As is well known in the medical arts, dosages for any one patient
depends upon many factors, including the patient's or subject's
size, body surface area, age, the particular compound to be
administered, sex, time and route of administration, general
health, and other drugs being administered concurrently.
Proteinaceous pharmaceutically active matter may be present in
amounts between 0.1-10 .mu.g/kg body weight per dose; however,
doses below or above this exemplary range are envisioned,
especially considering the aforementioned factors. For topical
administration as it is particularly preferred in the context of
the present invention, a pharmaceutically active matter suitable
for topical administration as defined herein further above and
below containing antibody concentrations of 0.1 to 10 mg/ml,
preferably of 0.5 to 5 mg/ml is particularly envisaged. This
corresponds to the ranges used in the Examples as exemplified
further below wherein the antibody in liquid solution (PBS) or
mixed 1:2 with creme at concentrations between 0.5 to 5 mg/ml has
been used which corresponds to 0.5 to 5 mg/g in PBS or a creme with
the same density of PBS.
Thus, preferably, the antibody or the antigen-binding fragment
thereof and/or the virostatic agent are included in an effective
amount. The term "effective amount" refers to an amount sufficient
to induce a detectable therapeutic response in the subject to which
the pharmaceutical composition is to be administered. In accordance
with the above, the content of the antibody in the pharmaceutical
composition is not limited as far as it is useful for treatment as
described above, but preferably contains 0.0000001-10% by weight
per total composition. Further, the antibody described herein is
preferably employed in a carrier. Generally, an appropriate amount
of a pharmaceutically acceptable salt is used in the carrier to
render the composition isotonic. Examples of the carrier include
but are not limited to saline, Ringer's solution and dextrose
solution. Preferably, acceptable excipients, carriers, or
stabilisers are non-toxic at the dosages and concentrations
employed, including buffers such as citrate, phosphate, and other
organic acids; salt-forming counter-ions, e.g. sodium and
potassium; low molecular weight (>10 amino acid residues)
polypeptides; proteins, e.g. serum albumin, or gelatine;
hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such
as histidine, glutamine, lysine, asparagine, arginine, or glycine;
carbohydrates including glucose, mannose, or dextrins;
monosaccharides; disaccharides; other sugars, e.g. sucrose,
mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA;
non-ionic surfactants, e.g. Tween, Pluronics or polyethylene
glycol; antioxidants including methionine, ascorbic acid and
tocopherol; and/or preservatives, e.g. octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, e.g. methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and
their formulations are described in greater detail in Remington's
Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.
Progress can be monitored by periodic assessment. The antibody,
antigen-binding fragment thereof or the pharmaceutical composition
of the invention are administered locally as defined above in
contrast to a systemic administration. Preparations for topical
administration have already been described above and include, inter
alia, sterile aqueous or non-aqueous solutions, suspensions, and
emulsions as well as creams and suppositories. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Preservatives and other additives may also be present such
as, for example, antimicrobials, anti-oxidants, chelating agents,
and inert gases and the like. Furthermore, the pharmaceutical
composition of the invention may comprise further agents depending
on the intended use of the pharmaceutical composition. Said agents
may be, e.g., Tween, EDTA, Citrate, Sucrose as well as other agents
being suitable for the intended use of the pharmaceutical
composition that are well-known to the person skilled in the
art.
In accordance with this invention, the term "pharmaceutical
composition" relates to a composition for administration to a
patient, preferably a human patient. In the context of the present
invention that medicament/pharmaceutical composition is to be
administered topically to a patient who suffers from an acute
infection of mucosal or epidermal tissue caused by HSV-1 or HSV-2
in accordance with the present invention. In the context of the
present invention, the subject, i.e., the patient refers to human
patient. Thus, the present invention also relates to a
pharmaceutical composition, comprising an effective amount of the
antibody or the antigen-binding fragment thereof for use in
treating an acute infection of mucosal or epidermal tissue in a
subject caused by HSV-1 or HSV-2 selected from the group consisting
of Herpes simplex labialis, Herpes simplex genitalis, chronic or
disseminated cutaneous herpes simplex infection, Herpes gladiatorum
and Eczema herpeticum and at least one pharmaceutically acceptable
excipient. As regards the preferred embodiments of the
pharmaceutical composition the same applies, mutatis mutandis, as
has been set forth above in the context of the anti-HSV antibody or
an antigen-binding fragment thereof for use in treating an acute
infection of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum wherein said antibody is to be topically administered as
well as the pharmaceutical composition as defined above.
The invention also relates to a method for the treatment of an
acute infection of mucosal or epidermal tissue in a subject caused
by HSV-1 or HSV-2 selected from the group consisting of Herpes
simplex labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum comprising the step of topically administering the
antibody or antigen-binding fragment as defined above. Thus, the
present invention relates to a method of the treatment of acute
infections of mucosal or epidermal tissue in a subject caused by
HSV-1 or HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum in a subject wherein the antibody or the antigen-binding
fragment thereof is administered topically to the subject in a
therapeutically effective amount. As regards the preferred
embodiments of the method for treatment the same applies, mutatis
mutandis, as has been set forth above in the context of the
anti-HSV antibody or an antigen-binding fragment thereof or the
pharmaceutical composition for use in treating an acute infection
of mucosal or epidermal tissue in a subject caused by HSV-1 or
HSV-2 selected from the group consisting of Herpes simplex
labialis, Herpes simplex genitalis, chronic or disseminated
cutaneous herpes simplex infection, Herpes gladiatorum and Eczema
herpeticum wherein said antibody is to be topically administered as
well as the pharmaceutical composition as defined above.
In the present invention, the subject is in a preferred embodiment
a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g.,
rat, mouse, and guinea pig, or a primate, e.g., gorilla,
chimpanzee, and human. In a most preferable embodiment, the subject
is a human.
Other aspects and advantages of the invention will be described in
the following examples, which are given for purposes of
illustration and not by way of limitation. Each publication,
patent, patent application or other document cited in this
application is hereby incorporated by reference in its
entirety.
FIG. 1: compares the survival of immunodeficient mice with acute
genital HSV-2 infection after topical treatment either with the
humanized monoclonal antibody hu2c or acyclovir. Female mice
(NOD.CB17-Prkdc.sup.scid/NCrHsd) were treated with a long-acting
progestin (Depo-Clinovir, Pharmacia) 7 days prior to viral
challenge to increase susceptibility to HSV-2 infection and to
eliminate differences caused by the estrous cycle. Anesthetized
mice were vaginally challenged with a lethal dose of
5.times.10.sup.5 PFU of HSV-2 G (20 .mu.l). Mice displaying visible
infection (perineal hair loss, reddening, swelling) were treated
one day after viral challenge (A) once with 40 .mu.l hu2c (5 mg/ml)
(.circle-solid.) or 40 .mu.l control IgG (5 mg/ml) (.quadrature.)
or (B) twice daily for 4 days with 40 .mu.l ACV (25 mg/ml)
(.DELTA.) or 40 .mu.l PBS (x). Drug solutions or PBS were applied
topically to the outer genital epithelium. Mice were monitored for
36 days after viral inoculation. Mice displaying sever systemic
signs and/or severe lesions/zoster were killed. Surviving mice were
sacrificed at day 36. Test groups contained eight animals each,
control groups contained five animals each. Kaplan-Meier survival
curves were analyzed by log-rank (Mantel-Cox) test. Two-tailed
significance tests were used to compare the significance level
between two groups. All protocols were approved by the Animal Care
and Use Committee.
FIG. 2: shows the clinical scoring of acute genital HSV-2 infection
after topical treatment with the humanized monoclonal antibody hu2c
or acyclovir. Mice displaying visible infection (perineal hair
loss, reddening, swelling) one day after intravaginal challenge
with a lethal dose of 5.times.105 PFU of HSV-2 G (20 .mu.l) were
treated (A & C) twice daily for 4 days with (A) 40 .mu.l PBS or
(C) 40 .mu.l ACV (25 mg/ml), or treated (B & D) once at 24 h
post infection with (B) 40 .mu.l hu2c (5 mg/ml) or (D) 40 .mu.l
control IgG (5 mg/ml). Drug solutions or PBS were applied topically
to the outer genital epithelium.
Infected animals were observed daily and their clinical status was
scored as follows: 0, lack of symptoms, no lesions; 1-2, redeness
and/or swelling (erosion); 3, localized lesion <1 mm; 4-5,
localized lesion 2-3 mm; 6-7 localized lesion 4-5 mm; 8-9, severe
hyperemia, destruction of the epithelium and stroma with necrosis;
10, systemic signs, death. Animals with grading >8 were killed
to prevent undue suffering. Test groups contained eight animals
each, control groups contained five animals each. Arrows indicate
time points of treatment.
EXAMPLES
Example 1: Topical Application of a Humanized Anti-HSV Antibody
1. Subjects, Materials, Methods
1.1 Generation and Production of a HSV Neutralizing Humanized
Monoclonal Antibody. Recently, it has been demonstrated that
cross-linking of a highly conserved glycoprotein B epitope of
HSV-1/2 through the murine monoclonal antibody mAb 2c does not only
result in highly efficient neutralization of free virions but also
in inhibition of direct virus spread from infected to non-infected
cells (Krawczyk A, et al., Journal of virology 2011;
85(4):1793-1803). To exploit these unique properties for
therapeutic use in humans, we generated a humanized derivative of
mAb 2c. For the vast majority of humanized antibodies retention of
a set of potentially immunogenic murine residues within the human
frameworks is usually required for maintaining the structural
integrity of the grafted antigen binding loops. In order to
generate a humanized antibody with the lowest possible immunogenic
potential any framework manipulations had been avoided by careful
selection of appropriate human germline sequences and simultaneous
employment of our previously described sequence multi-alignment
approach (Krauss J, et al., Protein Eng 2003; 16(10):753-759). To
identify appropriate human germline acceptor scaffolds for grafting
the mAb 2c complementarity determining regions (CDRs), variable
domain framework sequences of mAb 2c were aligned to corresponding
human sequences of the V Base database
(http://vbase.mrc-cpe.cam.ac.uk/). The highest framework sequence
identities to the corresponding murine mAb 2c variable light
(V.sub.L) and variable heavy (V.sub.H) chain sequence showed the
human germline sequences DP28 (88.5%) and DPK13 (88.9%),
respectively. Hence, CDR coding gene segments of the murine
donor-antibody 2c (i.e. 2c V.sub.L-CDR1/2/3 and 2c
V.sub.H-CDR1/2/3) were grafted into acceptor frameworks coding for
DP28 and DPK13, respectively. Variable domain encoding genes of the
chimeric and humanized V.sub.L chain and V.sub.H chain were
subsequently cloned into immunoglobulin expression vectors
containing a human constant heavy .gamma.1 chain, and a human
constant .kappa. chain, respectively. The humanized antibody was
either produced from stably transfected Sp2/0 mouse myeloma cell
lines or transient transfected HEK293 cells under serum-free
conditions and purified from culture supernatants to homogeneity by
protein A chromatography. Purity was assessed by gel filtration
chromatography (Superdex 200GL, GE Healthcare) as .gtoreq.95%
(Krawczyk A, et al., Proc Natl Acad Sci USA 2013;
110(17):6760-6765).
1.2 Trial Description Between 2010-2013, twelve healthy 30-59 year
old volunteers (7 female, 5 male) with an acute recurrence of oral
herpes infection (cold sores) were treated. Volunteers presented
themselves when the onset of initial HSV symptoms (itching of the
lips, burning or tingling near the lips or mouth area) occurred or
had progressed to visible skin disorders on the outer lips.
Observed skin disorders included small to large blisters filled
with clear yellowish fluid or external herpetic lesions including
leaking red blisters. Oral herpes infection of the mouth area is
mainly caused by the herpes simplex virus type 1 (HSV-1). However,
sometimes HSV-2 is spread to the mouth during oral sex, causing
oral herpes. The type of HSV infection (HSV-1 or HSV-2) was not
analyzed. The antibody was packaged as sterile solutions either in
PBS or PBS/ash creme (1:1) at concentrations of 0.7-1 mg/ml.
Participants applied approx. 10 .mu.l of the antibody topically
once, once per day for two days or for a total of three times
maximum.
2. Results ZOVIRAX Cream had been evaluated in 2 double-blind,
randomized, placebo (vehicle)-controlled trials (see Zovirax N-.
Zovirax Prescribing Information.
http://wwwaccessdatafdagov/drugsatfda_docs/label/2002/21478_zovirax_Ib|pd-
f#page=1&zoom=auto,0,792). In the Zovirax studies, subjects
were instructed to initiate treatment within 1 hour of noticing
signs or symptoms and continue treatment for 4 days, with
application of study medication 5 times per day. In both studies,
the mean duration of the recurrent herpes labialis episode was
approximately one-half day shorter in the subjects treated with
ZOVIRAX Cream (n=682) compared with subjects treated with placebo
(n=703) (approximately 4.5 days versus 5 days, respectively). No
significant difference was observed between subjects receiving
ZOVIRAX Cream or vehicle in the prevention of progression of cold
sore lesions. Compared to previous HSV outbreaks that have been
treated with aciclovir (Zovirax creme) all participants using the
antibody solution reported a fast symptom and pain relief within 24
h after application of the antibody. In contrast to the experiences
with aciclovir therapy active blisters regressed and did not turn
into weeping blisters when treated topically with the antibody.
When antibody treatment was started at the stage of visible
external herpetic lesions, participants reported a rapid healing
and disappearance of crusted areas. All participants reported in
contrast to their experience with Zovirax that the infected area
did not spread upon antibody treatment. One volunteer experienced
Herpes labialis at the upper and lower lip at the same time and
started antibody treatment for the upper lip (three times) and
Zovirax treatment for the lower lip (3-4 times a day for 3 days).
At time of treatment several small blisters were visible. For the
antibody treated HSV infection a quick recovery was observed.
Blisters of the upper lip disappeared within 24 h, the swelling
subsided within 48 h and no lesions occurred. The infection of
lower lip treated with Zovirax remained painful for 3 days,
blisters grew together into larger blister which eventually broke
open. The occurred lesions took two weeks to heal. Efficacy of the
treatment seemed to be independent from the antibody formulation
(PBS or PBS/ash creme). Interestingly the participants have the
impression that the overall rates of clinical reactivation tend to
be reduced.
Example 2: Topical Application of Anti-HSV mAb hu2c in Animal
Experiments
The ability of the humanized monoclonal antibody hu2c to alter the
clinical course of acute genital HSV-2 infection in immunodeficient
mice following a single topical treatment with 200 .mu.g mAb hu2c
(5 mg/ml) was investigated. To infect 100% of mice as assessed by
visible lesions and culture of vaginal lavage, a viral inoculum of
5.times.105 PFU of HSV-2 G was delivered to the vagina of
anesthetized mice. Although acute HSV-1 or HSV-2 infections result
in a fatal outcome in 100% of mice with severe combined
immunodeficiency when compared to 70-90% mortality in
immunocompetent mice, the immunodeficient model has nevertheless
been chosen to discriminate a possible clinical efficacy of the
therapy from an elimination of the viral infection due to immune
effector cells of the mouse (Minagawa et al., Arch Virol 103, 73-82
(1988); Nagafuchi et al., J Gen Virol 44, 715-723 (1979)). Within
genital mucosa the expansion rates of HSV-2 are extremely rapid. At
24 h after viral challenge infected mice received topically at the
infected area either twice per day for 4 days 1 mg acyclovir (ACV)
(25 mg/ml) or 40 .mu.l buffer (PBS) or a single treatment of 200
.mu.g mAb hu2c (5 mg/ml) or 200 .mu.g control mAb (5 mg/ml).
Clinical efficacy of the HSV-specific mAb was compared to the
irrelevant mAb (isotypcontrol), ACV and PBS treatment by means of
Kaplan-Meier survival curves and daily assessment of the clinical
status of the mucous membranes of the genital and anal area.
Results from mice displaying visible infection (perineal hair loss,
reddening, swelling) and detectable peripheral replication 24 h
after infection were evaluated. As expected, no significant
differences in overall survival were observed in control groups
treated either with an irrelevant mAb vs PBS and all mice were dead
by day 7 after infection (FIGS. 1A & B). Surprisingly, a single
topical application of mAb hu2c to the outer genital infected
epithelium resulted in a statistically significant difference in
survival curves (P=0.003) when compared to the control groups (FIG.
1 A) and even prevented the lethal outcome of the infection in one
mouse. Survival curves of the antibody hu2c treated group (FIG. 1
A) and the ACV treated group (FIG. 1 B) showed no significant
differences (P>0.5) although ACV was applied twice daily for 4
days. The medical advantage of the topical antibody therapy over
the standard therapy with ACV became even more apparent when
evaluating the clinical status of the acute genital infection over
a period of 14 days (FIG. 2). A clinical score grading can be
applied to investigate if the clinical course of an infection can
be altered upon treatment (Minagawa et al., Arch Virol 103, 73-82
(1988); Sanna et al., Virology 215, 101-106 (1996)). The clinical
status of vaginitis/vulvitis was scored as follows: 0, lack of
symptoms, no lesions; 1-2, redeness and/or swelling (erosion); 3,
localized lesion <1 mm; 4-5, localized lesion 2-3 mm; 6-7
localized lesion 4-5 mm; 8-9, severe hyperemia, destruction of the
epithelium and stroma with necrosis; 10, systemic signs, death.
Acute genital HSV-2 infection resolved in 7 out of 8 mice (88%)
within 48 h post single topical treatment with anti-HSV mAb hu2c
(FIG. 2D). In contrast, animals treated with ACV displayed an
extremely heterogeneous clinical grading. At 48 h under ongoing
treatment with ACV (twice per day, for 4 days) local genital
symptoms resolved only in 1 out of 8 mice (13%), and 72 h after
commencement of ACV treatment only 5 out of 8 mice (63%) had no
local signs (FIG. 2C). Although both, the hu2c antibody and ACV
were applied only topically, HSV-2 lethal encephalitis could be
prevented in 1 out of 8 mice in both cases. Mice either treated
with buffer or an irrelevant control mAb had progressive local
HSV-2 infections spreading across the genital and anal areas and
systemic dissemination of the virus resulted in the death of all
animals at day 7 (FIGS. 2 A & B).
SEQUENCE LISTINGS
1
1617PRTArtificial Sequence2cVH CDR1 1Thr Ser Gly Met Ser Val Gly1
5216PRTArtificial Sequence2cVH CDR2 2His Ile Trp Trp Asn Asn Asp
Lys Tyr Tyr Lys Pro Ala Leu Lys Ser1 5 10 15312PRTArtificial
Sequence2cVH CDR3 3Ile Tyr Tyr Gly Tyr Arg Pro Tyr Ala Met Asp Tyr1
5 10416PRTArtificial Sequence2cVL CDR1 4Arg Ser Ser Gln Ser Ile Val
His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 1557PRTArtificial
Sequence2cVL CDR2 5Lys Val Ser Asn Arg Phe Ser1 569PRTArtificial
Sequence2cVL CDR3 6Phe Gln Gly Ser His Val Pro Trp Ser1
57122PRTArtificial Sequencehuman germline VH
frameworkVARIANT(100)..(107)Xaa can be any naturally occurring
amino acid 7Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro
Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu
Ser Thr Ser 20 25 30Gly Met Arg Val Ser Trp Ile Arg Gln Pro Pro Gly
Lys Ala Leu Glu 35 40 45Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys
Phe Tyr Ser Thr Ser 50 55 60Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp
Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp
Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 1208114PRTArtificial Sequencehuman germline
VL framework 8Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Leu Asp Ser 20 25 30Asp Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln Lys Pro Gly Gln 35 40 45Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser
Tyr Arg Ala Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys65 70 75 80Ile Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95Arg Ile Glu Phe Pro Trp
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110Lys
Arg9122PRTArtificial SequenceVH of the humanized mAb hu2c antibody
9Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5
10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu 35 40 45Trp Leu Ala His Ile Trp Trp Asn Asn Asp Lys Tyr Tyr
Lys Pro Ala 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ile Tyr Tyr Gly Tyr Arg
Pro Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 12010113PRTArtificial SequenceVL of the humanized
mAb hu2c antibody 10Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Trp
Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110Arg11903PRTHSV1 strain F 11Met Arg Gln Gly Ala Ala Arg Gly Cys
Arg Trp Phe Val Val Trp Ala1 5 10 15Leu Leu Gly Leu Thr Leu Gly Val
Leu Val Ala Ser Ala Ala Pro Ser 20 25 30Ser Pro Gly Thr Pro Gly Val
Ala Ala Ala Thr Gln Ala Ala Asn Gly 35 40 45Gly Pro Ala Thr Pro Ala
Pro Pro Ala Pro Gly Pro Ala Pro Thr Gly 50 55 60Asp Thr Lys Pro Lys
Lys Asn Lys Lys Pro Lys Asn Pro Pro Pro Pro65 70 75 80Arg Pro Ala
Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu 85 90 95Arg Glu
His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe 100 105
110Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln
115 120 125Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr
Glu Gly 130 135 140Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr
Lys Phe Lys Ala145 150 155 160Thr Met Tyr Tyr Lys Asp Val Thr Val
Ser Gln Val Trp Phe Gly His 165 170 175Arg Tyr Ser Gln Phe Met Gly
Ile Phe Glu Asp Arg Ala Pro Val Pro 180 185 190Phe Glu Glu Val Ile
Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser 195 200 205Thr Ala Lys
Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg 210 215 220Asp
Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr225 230
235 240Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro
Ser 245 250 255Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn
Cys Ile Val 260 265 270Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr
Asp Glu Phe Val Leu 275 280 285Ala Thr Gly Asp Phe Val Tyr Met Ser
Pro Phe Tyr Gly Tyr Arg Glu 290 295 300Gly Ser His Thr Glu His Thr
Ser Tyr Ala Ala Asp Arg Phe Lys Gln305 310 315 320Val Asp Gly Phe
Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr 325 330 335Ala Pro
Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala 340 345
350Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp
355 360 365Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser
Phe Arg 370 375 380Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr
Asn Leu Thr Glu385 390 395 400Tyr Pro Leu Ser Arg Val Asp Leu Gly
Asp Cys Ile Gly Lys Asp Ala 405 410 415Arg Asp Ala Met Asp Arg Ile
Phe Ala Arg Arg Tyr Asn Ala Thr His 420 425 430Ile Lys Val Gly Gln
Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe Leu 435 440 445Ile Ala Tyr
Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val 450 455 460Arg
Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr Pro465 470
475 480Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys
Thr 485 490 495Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr
Asn His Ile 500 505 510Gln Arg His Val Asn Asp Met Leu Gly Arg Val
Ala Ile Ala Trp Cys 515 520 525Glu Leu Gln Asn His Glu Leu Thr Leu
Trp Asn Glu Ala Arg Lys Leu 530 535 540Asn Pro Asn Ala Ile Ala Ser
Ala Thr Val Gly Arg Arg Val Ser Ala545 550 555 560Arg Met Leu Gly
Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala 565 570 575Ala Asp
Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg Pro 580 585
590Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln
595 600 605Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu
Arg Leu 610 615 620Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His
Arg Arg Tyr Phe625 630 635 640Thr Phe Gly Gly Gly Tyr Val Tyr Phe
Glu Glu Tyr Ala Tyr Ser His 645 650 655Gln Leu Ser Arg Ala Asp Ile
Thr Thr Val Ser Thr Phe Ile Asp Leu 660 665 670Asn Ile Thr Met Leu
Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr 675 680 685Thr Arg His
Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val 690 695 700Gln
Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr705 710
715 720Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly
Ala 725 730 735Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly
Lys Val Val 740 745 750Met Gly Ile Val Gly Gly Val Val Ser Ala Val
Ser Gly Val Ser Ser 755 760 765Phe Met Ser Asn Pro Phe Gly Ala Leu
Ala Val Gly Leu Leu Val Leu 770 775 780Ala Gly Leu Ala Ala Ala Phe
Phe Ala Phe Arg Tyr Val Met Arg Leu785 790 795 800Gln Ser Asn Pro
Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu 805 810 815Lys Asn
Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly Gly 820 825
830Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg Tyr
835 840 845Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala
Lys Lys 850 855 860Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr
Asp Met Val Met865 870 875 880Arg Lys Arg Arg Asn Thr Asn Tyr Thr
Gln Val Pro Asn Lys Asp Gly 885 890 895Asp Ala Asp Glu Asp Asp Leu
90012904PRTHerpes Simplex Virus 1 strain KOS 12Met His Gln Gly Ala
Pro Ser Trp Gly Arg Arg Trp Phe Val Val Trp1 5 10 15Ala Leu Leu Gly
Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro 20 25 30Thr Ser Pro
Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn 35 40 45Gly Gly
Pro Ala Thr Pro Ala Pro Pro Pro Leu Gly Ala Ala Pro Thr 50 55 60Gly
Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro65 70 75
80Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr
85 90 95Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala
Asn 100 105 110Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val
Gln Phe Glu 115 120 125Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly
Gln Asn Tyr Thr Glu 130 135 140Gly Ile Ala Val Val Phe Lys Glu Asn
Ile Ala Pro Tyr Lys Phe Lys145 150 155 160Ala Thr Met Tyr Tyr Lys
Asp Val Thr Val Ser Gln Val Trp Phe Gly 165 170 175His Arg Tyr Ser
Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val 180 185 190Pro Phe
Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg 195 200
205Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His
210 215 220Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn
Ala Ala225 230 235 240Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp
Leu Lys Tyr Asn Pro 245 250 255Ser Arg Val Glu Ala Phe His Arg Tyr
Gly Thr Thr Val Asn Cys Ile 260 265 270Val Glu Glu Val Asp Ala Arg
Ser Val Tyr Pro Tyr Asp Glu Phe Val 275 280 285Leu Ala Thr Gly Asp
Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg 290 295 300Glu Gly Ser
His Thr Glu His Thr Thr Tyr Ala Ala Asp Arg Phe Lys305 310 315
320Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala
325 330 335Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe
Thr Val 340 345 350Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys
Thr Met Thr Lys 355 360 365Trp Gln Glu Val Asp Glu Met Leu Arg Ser
Glu Tyr Gly Gly Ser Phe 370 375 380Arg Phe Ser Ser Asp Ala Ile Ser
Thr Thr Phe Thr Thr Asn Leu Thr385 390 395 400Glu Tyr Pro Leu Ser
Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp 405 410 415Ala Arg Asp
Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr 420 425 430His
Ile Lys Val Gly Gln Pro Gln Tyr Tyr Gln Ala Asn Gly Gly Phe 435 440
445Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr
450 455 460Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn
Pro Thr465 470 475 480Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser
Val Glu Arg Ile Lys 485 490 495Thr Thr Ser Ser Ile Glu Phe Ala Arg
Leu Gln Phe Thr Tyr Asn His 500 505 510Ile Gln Arg His Val Asn Asp
Met Leu Gly Arg Val Ala Ile Ala Trp 515 520 525Cys Glu Leu Gln Asn
His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys 530 535 540Leu Asn Pro
Asn Ala Ile Ala Ser Val Thr Val Gly Arg Arg Val Ser545 550 555
560Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val
565 570 575Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser
Ser Arg 580 585 590Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe
Arg Tyr Glu Asp 595 600 605Gln Gly Pro Leu Val Glu Gly Gln Leu Gly
Glu Asn Asn Glu Leu Arg 610 615 620Leu Thr Arg Asp Ala Ile Glu Pro
Cys Thr Val Gly His Arg Arg Tyr625 630 635 640Phe Thr Phe Gly Gly
Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser 645 650 655His Gln Leu
Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp 660 665 670Leu
Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val 675 680
685Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu
690 695 700Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp
Ile Asp705 710 715 720Thr Val Ile His Ala Asp Ala Asn Ala Ala Met
Phe Ala Gly Leu Gly 725 730 735Ala Phe Phe Glu Gly Met Gly Asp Leu
Gly Arg Ala Val Gly Lys Val 740 745 750Val Met Gly Ile Val Gly Gly
Val Val Ser Ala Val Ser Gly Val Ser 755 760 765Ser Phe Met Ser Asn
Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val 770 775 780Leu Ala Gly
Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg785 790 795
800Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu
805 810 815Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu
Glu Gly 820 825 830Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg
Glu Met Ile Arg 835 840 845Tyr Met Ala Leu Val Ser Ala Met Glu Arg
Thr Glu His Lys Ala Lys 850 855 860Lys Lys Gly Thr Ser Ala Leu Leu
Ser Ala Lys Val Thr Asp Met Val865 870 875 880Met Arg Lys Arg Arg
Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp 885 890 895Gly Asp Ala
Asp Glu Asp Asp Leu 90013904PRTHerpes Simplex Virus 1 strain
gC-39-R6 13Met His Gln Gly Ala Pro Ser Trp Gly Arg Arg Trp Phe Val
Val Trp1 5 10 15Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser
Ala Ala Pro 20 25 30Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr
Gln Ala Ala Asn 35 40
45Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr
50 55 60Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr
Pro65 70 75 80Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly
His Ala Thr 85 90 95Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn
Thr Asp Ala Asn 100 105 110Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala
Thr Val Val Gln Phe Glu 115 120 125Gln Pro Arg Arg Cys Pro Thr Arg
Pro Glu Gly Gln Asn Tyr Thr Glu 130 135 140Gly Ile Ala Val Val Phe
Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys145 150 155 160Ala Thr Met
Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly 165 170 175His
Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val 180 185
190Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg
195 200 205Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala
Phe His 210 215 220Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro
Ala Asn Ala Ala225 230 235 240Thr Arg Thr Ser Arg Gly Trp His Thr
Thr Asp Leu Lys Tyr Asn Pro 245 250 255Ser Arg Val Glu Ala Phe His
Arg Tyr Gly Thr Thr Val Asn Cys Ile 260 265 270Val Glu Glu Val Asp
Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val 275 280 285Leu Ala Thr
Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg 290 295 300Glu
Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys305 310
315 320Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg
Ala 325 330 335Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys
Phe Thr Val 340 345 350Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val
Cys Thr Met Thr Lys 355 360 365Trp Gln Glu Val Asp Glu Met Leu Arg
Ser Glu Tyr Gly Gly Ser Phe 370 375 380Arg Phe Ser Ser Asp Ala Ile
Ser Thr Thr Phe Thr Thr Asn Leu Thr385 390 395 400Glu Tyr Pro Leu
Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp 405 410 415Ala Arg
Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr 420 425
430His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe
435 440 445Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu
Leu Tyr 450 455 460Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro
Pro Asn Pro Thr465 470 475 480Pro Pro Pro Pro Gly Ala Ser Ala Asn
Ala Ser Val Glu Arg Ile Lys 485 490 495Thr Thr Ser Ser Ile Glu Phe
Ala Arg Leu Gln Phe Thr Tyr Asn His 500 505 510Ile Gln His His Val
Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp 515 520 525Cys Glu Leu
Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys 530 535 540Leu
Asn Pro Asn Ala Ile Ala Ser Val Thr Val Gly Arg Arg Val Ser545 550
555 560Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro
Val 565 570 575Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile
Ser Ser Arg 580 585 590Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser
Phe Arg Tyr Glu Asp 595 600 605Gln Gly Pro Leu Val Glu Gly Gln Leu
Gly Glu Asn Asn Glu Leu Arg 610 615 620Leu Thr Arg Asp Ala Ile Glu
Pro Cys Thr Val Gly His Arg Arg Tyr625 630 635 640Phe Thr Phe Gly
Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser 645 650 655His Gln
Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp 660 665
670Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val
675 680 685Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr
Thr Glu 690 695 700Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe
Ala Asp Ile Asp705 710 715 720Thr Val Ile His Ala Asp Ala Asn Ala
Ala Met Phe Ala Gly Leu Gly 725 730 735Ala Phe Phe Glu Gly Met Gly
Asp Leu Gly Arg Ala Val Gly Lys Val 740 745 750Val Met Gly Ile Val
Gly Gly Val Val Ser Ala Val Ser Gly Val Ser 755 760 765Ser Phe Met
Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val 770 775 780Leu
Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg785 790
795 800Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys
Glu 805 810 815Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly
Glu Glu Gly 820 825 830Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala
Arg Glu Met Ile Arg 835 840 845Tyr Met Ala Leu Val Ser Ala Met Glu
Arg Thr Glu His Lys Ala Lys 850 855 860Lys Lys Gly Thr Ser Ala Leu
Leu Ser Ala Lys Val Thr Asp Met Val865 870 875 880Met Arg Lys Arg
Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp 885 890 895Gly Asp
Ala Asp Glu Asp Asp Leu 90014904PRTHerpes Simplex Virus 2 strain
HG52 14Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu
Val1 5 10 15Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro
Arg Ala 20 25 30Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly
Pro Ala Ser 35 40 45Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys
Ala Arg Lys Arg 50 55 60Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala
Thr Pro Pro Pro Asp65 70 75 80Ala Asn Ala Thr Val Ala Ala Gly His
Ala Thr Leu Arg Ala His Leu 85 90 95Arg Glu Ile Lys Val Glu Asn Ala
Asp Ala Gln Phe Tyr Val Cys Pro 100 105 110Pro Pro Thr Gly Ala Thr
Val Val Gln Phe Glu Gln Pro Arg Arg Cys 115 120 125Pro Thr Arg Pro
Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val 130 135 140Phe Lys
Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr145 150 155
160Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln
165 170 175Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu
Glu Val 180 185 190Ile Asp Lys Ile Asn Thr Lys Gly Val Cys Arg Ser
Thr Ala Lys Tyr 195 200 205Val Arg Asn Asn Met Glu Thr Thr Ala Phe
His Arg Asp Asp His Glu 210 215 220Thr Asp Met Glu Leu Lys Pro Ala
Lys Val Ala Thr Arg Thr Ser Arg225 230 235 240Gly Trp His Thr Thr
Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala 245 250 255Phe His Arg
Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp 260 265 270Ala
Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp 275 280
285Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr
290 295 300Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp
Gly Phe305 310 315 320Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala
Thr Ser Pro Thr Thr 325 330 335Arg Asn Leu Leu Thr Thr Pro Lys Phe
Thr Val Ala Trp Asp Trp Val 340 345 350Pro Lys Arg Pro Ala Val Cys
Thr Met Thr Lys Trp Gln Glu Val Asp 355 360 365Glu Met Leu Arg Ala
Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp 370 375 380Ala Ile Ser
Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Ser Leu Ser385 390 395
400Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile
405 410 415Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys
Val Gly 420 425 430Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu
Ile Ala Tyr Gln 435 440 445Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu
Tyr Val Arg Glu Tyr Met 450 455 460Arg Glu Gln Asp Arg Lys Pro Arg
Asn Ala Thr Pro Ala Pro Leu Arg465 470 475 480Glu Ala Pro Ser Ala
Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser 485 490 495Ser Ile Glu
Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg 500 505 510His
Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu 515 520
525Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro
530 535 540Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala
Arg Met545 550 555 560Leu Gly Asp Val Met Ala Val Ser Thr Cys Val
Pro Val Ala Pro Asp 565 570 575Asn Val Ile Val Gln Asn Ser Met Arg
Val Ser Ser Arg Pro Gly Thr 580 585 590Cys Tyr Ser Arg Pro Leu Val
Ser Phe Arg Tyr Glu Asp Gln Gly Pro 595 600 605Leu Ile Glu Gly Gln
Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg 610 615 620Asp Ala Leu
Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Ile Phe625 630 635
640Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu
645 650 655Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu
Asn Ile 660 665 670Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu
Val Tyr Thr Arg 675 680 685His Glu Ile Lys Asp Ser Gly Leu Leu Asp
Tyr Thr Glu Val Gln Arg 690 695 700Arg Asn Gln Leu His Asp Leu Arg
Phe Ala Asp Ile Asp Thr Val Ile705 710 715 720Arg Ala Asp Ala Asn
Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe 725 730 735Glu Gly Met
Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly 740 745 750Val
Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met 755 760
765Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly
770 775 780Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu
Gln Arg785 790 795 800Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr
Lys Glu Leu Lys Thr 805 810 815Ser Asp Pro Gly Gly Val Gly Gly Glu
Gly Glu Glu Gly Ala Glu Gly 820 825 830Gly Gly Phe Asp Glu Ala Lys
Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845Tyr Met Ala Leu Val
Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg 850 855 860Lys Lys Gly
Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn Met Val865 870 875
880Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp
885 890 895Glu Ala Gly Asp Glu Asp Glu Leu 90015904PRTHerpes
Simplex Virus 2 strain 333 15Met Arg Gly Gly Gly Leu Ile Cys Ala
Leu Val Val Gly Ala Leu Val1 5 10 15Ala Ala Val Ala Ser Ala Ala Pro
Ala Ala Pro Ala Ala Pro Arg Ala 20 25 30Ser Gly Gly Val Ala Ala Thr
Val Ala Ala Asn Gly Gly Pro Ala Ser 35 40 45Arg Pro Pro Pro Val Pro
Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg 50 55 60Lys Thr Lys Lys Pro
Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp65 70 75 80Ala Asn Ala
Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu 85 90 95Arg Glu
Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro 100 105
110Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys
115 120 125Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala
Val Val 130 135 140Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala
Thr Met Tyr Tyr145 150 155 160Lys Asp Val Thr Val Ser Gln Val Trp
Phe Gly His Arg Tyr Ser Gln 165 170 175Phe Met Gly Ile Phe Glu Asp
Arg Ala Pro Val Pro Phe Glu Glu Val 180 185 190Ile Asp Lys Ile Asn
Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr 195 200 205Val Arg Asn
Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu 210 215 220Thr
Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg225 230
235 240Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu
Ala 245 250 255Phe His Arg Tyr Gly Thr Thr Val Thr Cys Ile Val Glu
Glu Val Asp 260 265 270Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val
Leu Ala Thr Gly Asp 275 280 285Phe Val Tyr Met Ser Pro Phe Tyr Gly
Tyr Arg Glu Gly Ser His Thr 290 295 300Glu His Thr Ser Tyr Ala Ala
Asp Arg Phe Lys Gln Val Asp Gly Phe305 310 315 320Tyr Ala Arg Asp
Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr 325 330 335Arg Asn
Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val 340 345
350Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp
355 360 365Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser
Ser Asp 370 375 380Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln
Tyr Ser Leu Ser385 390 395 400Arg Val Asp Leu Gly Asp Cys Ile Gly
Arg Asp Ala Arg Glu Ala Ile 405 410 415Asp Arg Met Phe Ala Arg Lys
Tyr Asn Ala Thr His Ile Lys Val Gly 420 425 430Gln Pro Gln Tyr Tyr
Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln 435 440 445Pro Leu Leu
Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met 450 455 460Arg
Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg465 470
475 480Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr
Ser 485 490 495Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His
Ile Gln Arg 500 505 510His Val Asn Asp Met Leu Gly Arg Ile Ala Val
Ala Trp Cys Glu Leu 515 520 525Gln Asn His Glu Leu Thr Leu Trp Asn
Glu Ala Arg Lys Leu Asn Pro 530 535 540Asn Ala Ile Ala Ser Ala Thr
Val Gly Arg Arg Val Ser Ala Arg Met545 550 555 560Leu Gly Asp Val
Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp 565 570 575Asn Val
Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr 580 585
590Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro
595 600 605Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu
Thr Arg 610 615 620Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg
Tyr Phe Ile Phe625 630 635 640Gly Gly Gly Tyr Val Tyr Phe Glu Glu
Tyr Ala Tyr Ser His Gln Leu 645 650 655Ser Arg Ala Asp Val Thr Thr
Val Ser Thr Phe Ile Asp Leu Asn Ile 660 665 670Thr Met Leu Glu Asp
His Glu Phe Val Pro Leu Gly Val Tyr Thr Arg 675 680 685His Glu Ile
Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg 690 695
700Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val
Ile705 710 715 720Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu
Cys Ala Phe Phe 725 730 735Glu Gly Met Gly Asp Leu Gly Arg Ala Val
Gly Lys Val Val Met Gly 740 745 750Val Val Gly Gly Val Val Ser Ala
Val Ser Gly Val Ser Ser Phe Met 755 760 765Ser Asn Pro Phe Gly Ala
Leu Ala Val Gly Leu Leu Val Leu Ala Gly 770 775 780Leu Val Ala Ala
Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg785 790 795 800Asn
Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr 805 810
815Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly
820 825 830Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met
Ile Arg 835 840 845Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu
His Lys Ala Arg 850 855 860Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser
Lys Val Thr Asn Met Val865 870 875 880Leu Arg Lys Arg Asn Lys Ala
Arg Tyr Ser Pro Leu His Asn Glu Asp 885 890 895Glu Ala Gly Asp Glu
Asp Glu Leu 90016901PRTHerpes Simplex Virus 2 strain MMA 16Met Arg
Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val1 5 10 15Ala
Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Arg Ala Ser Gly Gly 20 25
30Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser Arg Pro Pro
35 40 45Pro Val Pro Ser Pro Ala Thr Thr Arg Ala Arg Lys Arg Lys Thr
Lys 50 55 60Lys Pro Pro Glu Arg Pro Glu Ala Thr Pro Pro Pro Asp Ala
Asn Ala65 70 75 80Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His
Leu Arg Glu Ile 85 90 95Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val
Cys Pro Pro Pro Thr 100 105 110Gly Ala Thr Val Val Gln Phe Glu Gln
Pro Arg Arg Cys Pro Thr Arg 115 120 125Pro Glu Gly Gln Asn Tyr Thr
Glu Gly Ile Ala Val Val Phe Lys Glu 130 135 140Asn Ile Ala Pro Tyr
Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val145 150 155 160Thr Val
Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln Phe Met Gly 165 170
175Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu Glu Val Ile Asp Lys
180 185 190Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr Val
Arg Asn 195 200 205Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His
Glu Thr Asp Met 210 215 220Glu Leu Lys Pro Ala Lys Val Ala Thr Arg
Thr Ser Arg Gly Trp His225 230 235 240Thr Thr Asp Leu Lys Tyr Asn
Pro Ser Arg Val Glu Ala Phe His Arg 245 250 255Tyr Gly Thr Thr Val
Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser 260 265 270Val Tyr Pro
Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr 275 280 285Met
Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr 290 295
300Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe Tyr Ala
Arg305 310 315 320Asp Leu Thr Thr Lys Ala Gln Ala Thr Ser Pro Thr
Thr Arg Asn Leu 325 330 335Leu Thr Thr Pro Lys Phe Thr Val Ala Trp
Asp Trp Val Pro Lys Arg 340 345 350Pro Ala Val Cys Thr Met Thr Lys
Trp Gln Glu Val Asp Glu Met Leu 355 360 365Arg Ala Glu Tyr Gly Gly
Ser Phe Arg Phe Ser Ser Asp Ala Ile Ser 370 375 380Thr Thr Phe Thr
Thr Asn Leu Thr Glu Tyr Ser Leu Ser Arg Val Asp385 390 395 400Leu
Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile Asp Arg Met 405 410
415Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln
420 425 430Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln Pro
Leu Leu 435 440 445Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr
Met Arg Glu Gln 450 455 460Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala
Pro Leu Arg Glu Ala Pro465 470 475 480Ser Ala Asn Ala Ser Val Glu
Arg Ile Lys Thr Thr Ser Ser Ile Glu 485 490 495Phe Ala Arg Leu Gln
Phe Thr Tyr Asn His Ile Gln Arg His Val Asn 500 505 510Asp Met Leu
Gly Arg Ile Ala Val Ala Trp Cys Glu Leu Gln Asn His 515 520 525Glu
Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile 530 535
540Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly
Asp545 550 555 560Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro
Asp Asn Val Ile 565 570 575Val Gln Asn Ser Met Arg Val Ser Ser Arg
Pro Gly Thr Cys Tyr Ser 580 585 590Arg Pro Leu Val Ser Phe Arg Tyr
Glu Asp Gln Gly Pro Leu Ile Glu 595 600 605Gly Gln Leu Gly Glu Asn
Asn Glu Leu Arg Leu Thr Arg Asp Ala Leu 610 615 620Glu Pro Cys Thr
Val Gly His Arg Arg Tyr Phe Ile Phe Gly Gly Gly625 630 635 640Tyr
Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu Ser Arg Ala 645 650
655Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met Leu
660 665 670Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His
Glu Ile 675 680 685Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln
Arg Arg Asn Gln 690 695 700Leu His Asp Leu Arg Phe Ala Asp Ile Asp
Thr Val Ile Arg Ala Asp705 710 715 720Ala Asn Ala Ala Met Phe Ala
Gly Leu Cys Ala Phe Phe Glu Gly Met 725 730 735Gly Asp Leu Gly Arg
Ala Val Gly Lys Val Val Met Gly Val Val Gly 740 745 750Gly Val Val
Ser Ala Val Ser Gly Val Ser Ser Phe Met Ser Asn Pro 755 760 765Phe
Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly Leu Val Ala 770 775
780Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg Asn Pro
Met785 790 795 800Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys
Thr Ser Asp Pro 805 810 815Gly Gly Val Gly Gly Glu Gly Glu Glu Gly
Ala Glu Gly Gly Gly Phe 820 825 830Asp Glu Ala Lys Leu Ala Glu Ala
Arg Glu Met Ile Arg Tyr Met Ala 835 840 845Leu Val Ser Ala Met Glu
Arg Thr Glu His Lys Ala Arg Lys Lys Gly 850 855 860Thr Ser Ala Leu
Leu Ser Ser Lys Val Thr Asn Met Val Leu Arg Lys865 870 875 880Arg
Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp Glu Ala Gly 885 890
895Asp Glu Asp Glu Leu 900
* * * * *
References